Variants derived from actriib and uses therefor

ABSTRACT

In certain aspects, the present invention provides compositions and methods for modulating (promoting or inhibiting) growth of a tissue, such as bone, cartilage, muscle, fat, and/or neuronal tissue. The present invention also provides methods of screening compounds that modulate activity of an ActRIIB protein and/or an ActRIIB ligand. The compositions and methods provided herein are useful in treating diseases associated with abnormal activity of an ActRIIB protein and/or an ActRIIB ligand.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/287,531, filed Feb. 27, 2019 (now pending), which is a continuationof U.S. application Ser. No. 15/201,031, filed Jul. 1, 2016 (now U.S.Pat. No. 10,259,861), which is a continuation of U.S. application Ser.No. 13/730,418, filed Dec. 28, 2012 (now U.S. Pat. No. 9,399,669), whichis a continuation of U.S. application Ser. No. 12/893,976, filed Sep.29, 2010 (now U.S. Pat. No. 8,343,933), which is a continuation of U.S.application Ser. No. 12/012,652, filed Feb. 4, 2008 (now U.S. Pat. No.7,842,663), which claims the benefit of U.S. Provisional ApplicationSer. No. 60/899,304, filed Feb. 2, 2007; 60/927,088 filed May 1, 2007;and 60/931,880, filed May 25, 2007. The specifications of each of theforegoing applications are incorporated herein by reference in theirentirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing, which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on Jan. 18, 2022 is named1848179-0002-024-106_Seq.txt and is 35,569 bytes in size.

BACKGROUND OF THE INVENTION

The transforming growth factor-beta (TGF-beta) superfamily contains avariety of growth factors that share common sequence elements andstructural motifs. These proteins are known to exert biological effectson a large variety of cell types in both vertebrates and invertebrates.Members of the superfamily perform important functions during embryonicdevelopment in pattern formation and tissue specification and caninfluence a variety of differentiation processes, includingadipogenesis, myogenesis, chondrogenesis, cardiogenesis, hematopoiesis,neurogenesis, and epithelial cell differentiation. The family is dividedinto two general branches: the BMP/GDF and the TGF-beta/Activin/BMP10branches, whose members have diverse, often complementary effects. Bymanipulating the activity of a member of the TGF-beta family, it isoften possible to cause significant physiological changes in anorganism. For example, the Piedmontese and Belgian Blue cattle breedscarry a loss-of-function mutation in the GDF8 (also called myostatin)gene that causes a marked increase in muscle mass. Grobet et al., NatGenet. 1997, 17(1):71-4. Furthermore, in humans, inactive alleles ofGDF8 are associated with increased muscle mass and, reportedly,exceptional strength. Schuelke et al., N Engl J Med 2004, 350:2682-8.

Changes in muscle, bone, cartilage and other tissues may be achieved byagonizing or antagonizing signaling that is mediated by an appropriateTGF-beta family member. Thus, there is a need for agents that functionas potent regulators of TGF-beta signaling.

SUMMARY OF THE INVENTION

In certain aspects, the present disclosure provides ActRIIBpolypeptides, particularly ActRIIB variants, including amino- andcarboxy-terminal truncations and sequence alterations. Such ActRIIBpolypeptides may be used for the treatment of a variety of disorders orconditions, in particular, muscle and neuromuscular disorders (e.g.,muscular dystrophy, amyotrophic lateral sclerosis (ALS), and muscleatrophy), adipose tissue disorders (e.g., obesity), metabolic disorders(e.g., type 2 diabetes), neurodegenerative disorders, and muscle wastingassociated with old age (sarcopenia), prostate cancer therapy, andcancer cachexia. In specific embodiments, ActRIIB polypeptides (e.g.,soluble ActRIIB polypeptides) can antagonize an ActRIIB receptor in anyprocess associated with ActRIIB activity. Optionally, ActRIIBpolypeptides of the invention may be designed to preferentiallyantagonize one or more ligands of ActRIIB receptors, such as GDF8 (alsocalled myostatin), GDF11, activin A, activin B, activin AB, Nodal, andBMP7 (also called OP-1), and may therefore be useful in the treatment ofadditional disorders. Examples of ActRIIB polypeptides include thenaturally occurring ActRIIB polypeptides as well as functional variantsthereof. The disclosure also provides a set of variants derived fromActRIIB that have greatly diminished affinity for activin whileretaining binding to GDF11. These variants exhibit desirable effects onmuscle while reducing effects on other tissues.

In certain aspects, the disclosure provides pharmaceutical preparationscomprising a soluble ActRIIB polypeptide that binds to an ActRIIB ligandsuch as GDF8, GDF11, activin, BMP7 or nodal, and a pharmaceuticallyacceptable carrier. Optionally, the soluble ActRIIB polypeptide binds toan ActRIIB ligand with a Kd less than 10 micromolar or less than 1micromolar, 100, 10 or 1 nanomolar. Optionally, the soluble ActRIIBpolypeptide inhibits ActRIIB signaling, such as intracellular signaltransduction events triggered by an ActRIIB ligand. A soluble ActRIIBpolypeptide for use in such a preparation may be any of those disclosedherein, such as a polypeptide having an amino acid sequence selectedfrom SEQ ID NOs: 1, 2, 5, 6 and 12, or having an amino acid sequencethat is at least 80%, 85%, 90%, 95%, 97% or 99% identical to an aminoacid sequence selected from SEQ ID NOs: 1, 2, 5, 6 and 12. A solubleActRIIB polypeptide may include a functional fragment of a naturalActRIIB polypeptide, such as one comprising at least 10, 20 or 30 aminoacids of a sequence selected from SEQ ID NOs: 1, 2, 5, 6 and 12 or asequence of SEQ ID NO: 1, lacking the C-terminal 1, 2, 3, 4, 5 or 10 to15 amino acids and lacking 1, 2, 3, 4 or 5 amino acids at theN-terminus. A preferred polypeptide will comprise a truncation relativeto SEQ ID NO:1 of between 2 and 5 amino acids at the N-terminus and nomore than 3 amino acids at the C-terminus. Another preferred polypeptideis that presented as SEQ ID NO:12. A soluble ActRIIB polypeptide mayinclude one or more alterations in the amino acid sequence (e.g., in theligand-binding domain) relative to a naturally occurring ActRIIBpolypeptide. The alteration in the amino acid sequence may, for example,alter glycosylation of the polypeptide when produced in a mammalian,insect or other eukaryotic cell or alter proteolytic cleavage of thepolypeptide relative to the naturally occurring ActRIIB polypeptide. Asoluble ActRIIB polypeptide may be a fusion protein that has, as onedomain, an ActRIIB polypeptide (e.g., a ligand-binding domain of anActRIIB or a variant thereof) and one or more additional domains thatprovide a desirable property, such as improved pharmacokinetics, easierpurification, targeting to particular tissues, etc. For example, adomain of a fusion protein may enhance one or more of in vivo stability,in vivo half life, uptake/administration, tissue localization ordistribution, formation of protein complexes, multimerization of thefusion protein, and/or purification. A soluble ActRIIB fusion proteinmay include an immunoglobulin Fc domain (wild-type or mutant) or a serumalbumin. In certain embodiments, an ActRIIB-Fc fusion comprises arelatively unstructured linker positioned between the Fc domain and theextracellular ActRIIB domain. This unstructured linker may correspond tothe roughly 15 amino acid unstructured region at the C-terminal end ofthe extracellular domain of ActRIIB (the “tail”), or it may be anartificial sequence of between 5 and 15, 20, 30, 50 or more amino acidsthat are relatively free of secondary structure. A linker may be rich inglycine and proline residues and may, for example, contain repeatingsequences of threonine/serine and glycines (e.g., TG₄ or SG₄ repeats). Afusion protein may include a purification subsequence, such as anepitope tag, a FLAG tag, a polyhistidine sequence, and a GST fusion.Optionally, a soluble ActRIIB polypeptide includes one or more modifiedamino acid residues selected from: a glycosylated amino acid, aPEGylated amino acid, a farnesylated amino acid, an acetylated aminoacid, a biotinylated amino acid, an amino acid conjugated to a lipidmoiety, and an amino acid conjugated to an organic derivatizing agent. Apharmaceutical preparation may also include one or more additionalcompounds such as a compound that is used to treat an ActRIIB-associateddisorder. Preferably, a pharmaceutical preparation is substantiallypyrogen free. In general, it is preferable that an ActRIIB protein beexpressed in a mammalian cell line that mediates suitably naturalglycosylation of the ActRIIB protein so as to diminish the likelihood ofan unfavorable immune response in a patient. Human and CHO cell lineshave been used successfully, and it is expected that other commonmammalian expression vectors will be useful.

In certain aspects, the disclosure provides packaged pharmaceuticalscomprising a pharmaceutical preparation described herein and labeled foruse in promoting growth of a tissue or diminishing or preventing a lossof a tissue in a human. Exemplary tissues include bone, cartilage,muscle, fat, and neuronal tissue.

In certain aspects, the disclosure provides soluble ActRIIB polypeptidescomprising an altered ligand-binding (e.g., GDF8-binding) domain. Suchaltered ligand-binding domains of an ActRIIB receptor comprise one ormore mutations at amino acid residues such as E37, E39, R40, K55, R56,Y60, A64, K74, W78, L79, D80, F82 and F101 of human ActRIIB. (Numberingis relative to SEQ ID NO:2). Optionally, the altered ligand-bindingdomain can have increased selectivity for a ligand such as GDF8/GDF11relative to a wild-type ligand-binding domain of an ActRIIB receptor. Toillustrate, these mutations are demonstrated herein to increase theselectivity of the altered ligand-binding domain for GDF11 (andtherefore, presumably, GDF8) over activin (presented with respect toActRIIB): K74Y, K74F, K741 and D801. The following mutations have thereverse effect, increasing the ratio of activin binding over GDF11:D54A, K55A, L79A and F82A. The overall (GDF11 and activin) bindingactivity can be increased by inclusion of the “tail” region or,presumably, an unstructured linker region, and also by use of a K74Amutation. Other mutations that caused an overall decrease in ligandbinding affinity, include: R40A, E37A, R56A, W78A, D80K, D80R, D80A,D80G, D80F, D80M and D80N. Mutations may be combined to achieve desiredeffects. For example, many of the mutations that affect the ratio ofGDF11:Activin binding have an overall negative effect on ligand binding,and therefore, these may be combined with mutations that generallyincrease ligand binding to produce an improved binding protein withligand selectivity.

Optionally, the altered ligand-binding domain has a ratio of K_(d) foractivin binding to K_(d) for GDF8 binding that is at least 2, 5, 10, oreven 100 fold greater relative to the ratio for the wild-typeligand-binding domain. Optionally, the altered ligand-binding domain hasa ratio of IC₅₀ for inhibiting activin to IC₅₀ for inhibiting GDF8/GDF11that is at least 2, 5, 10, or even 100 fold greater relative to thewild-type ligand-binding domain. Optionally, the altered ligand-bindingdomain inhibits GDF8/GDF11 with an IC₅₀ at least 2, 5, 10, or even 100times less than the IC₅₀ for inhibiting activin. These soluble ActRIIBpolypeptides can be fusion proteins that include an immunoglobulin Fcdomain (either wild-type or mutant). In certain cases, the subjectsoluble ActRIIB polypeptides are antagonists (inhibitors) of GDF8/GDF11.

Other variants of ActRIIB are contemplated, such as the following. Avariant ActRIIB fusion protein comprising a portion derived from theActRIIB sequence of SEQ ID NO:2 and a second polypeptide portion,wherein the portion derived from ActRIIB corresponds to a sequencebeginning at any of amino acids 21-29 of SEQ ID NO:2 (optionallybeginning at 22-25 of SEQ ID NO:2) and ending at any of amino acids109-134 of SEQ ID NO:2, and wherein the ActRIIB fusion protein inhibitssignaling by activin, myostatin and/or GDF11 in a cell-based assay. Thevariant ActRIIB fusion protein above, wherein the portion derived fromActRIIB corresponds to a sequence beginning at any of amino acids 20-29of SEQ ID NO:2 (optionally beginning at 22-25 of SEQ ID NO:2) and endingat any of amino acids 109-133 of SEQ ID NO:2. The variant ActRIIB fusionprotein above, wherein the portion derived from ActRIIB corresponds to asequence beginning at any of amino acids 20-24 of SEQ ID NO:2(optionally beginning at 22-25 of SEQ ID NO:2) and ending at any ofamino acids 109-133 of SEQ ID NO:2. The variant ActRIIB fusion proteinabove, wherein the portion derived from ActRIIB corresponds to asequence beginning at any of amino acids 21-24 of SEQ ID NO:2 and endingat any of amino acids 109-134 of SEQ ID NO:2. The variant ActRIIB fusionprotein above, wherein the portion derived from ActRIIB corresponds to asequence beginning at any of amino acids 20-24 of SEQ ID NO:2 and endingat any of amino acids 118-133 of SEQ ID NO:2. The variant ActRIIB fusionprotein above, wherein the portion derived from ActRIIB corresponds to asequence beginning at any of amino acids 21-24 of SEQ ID NO:2 and endingat any of amino acids 118-134 of SEQ ID NO:2. The variant ActRIIB fusionprotein above, wherein the portion derived from ActRIIB corresponds to asequence beginning at any of amino acids 20-24 of SEQ ID NO:2 and endingat any of amino acids 128-133 of SEQ ID NO:2. The variant ActRIIB fusionprotein above, wherein the portion derived from ActRIIB corresponds to asequence beginning at any of amino acids 20-24 of SEQ ID NO:2 and endingat any of amino acids 128-133 of SEQ ID NO:2. The variant ActRIIB fusionprotein above, wherein the portion derived from ActRIIB corresponds to asequence beginning at any of amino acids 21-29 of SEQ ID NO:2 and endingat any of amino acids 118-134 of SEQ ID NO:2. The variant ActRIIB fusionprotein above, wherein the portion derived from ActRIIB corresponds to asequence beginning at any of amino acids 20-29 of SEQ ID NO:2 and endingat any of amino acids 118-133 of SEQ ID NO:4. The variant ActRIIB fusionprotein above, wherein the portion derived from ActRIIB corresponds to asequence beginning at any of amino acids 21-29 of SEQ ID NO:2 and endingat any of amino acids 128-134 of SEQ ID NO:2. The variant ActRIIB fusionprotein above, wherein the portion derived from ActRIIB corresponds to asequence beginning at any of amino acids 20-29 of SEQ ID NO:2 and endingat any of amino acids 128-133 of SEQ ID NO:2. Surprisingly, constructsbeginning at 22-25 of SEQ ID NO:2 have activity levels greater thanproteins having the full extracellular domain of human ActRIIB. Any ofthe above variant ActRIIB fusion protein may be produced as a homodimer.Any of the above ActRIIB fusion proteins may have a heterologous portionthat comprises a constant region from an IgG heavy chain, such as an Fcdomain.

Other variant ActRIIB proteins are contemplated, such as the following.A variant ActRIIB protein comprising an amino acid sequence that is atleast 80% identical to the sequence of amino acids 29-109 of SEQ ID NO:2, wherein the position corresponding to 64 of SEQ ID NO:2 is an R or K,and wherein the variant ActRIIB protein inhibits signaling by activin,myostatin and/or GDF11 in a cell-based assay. The variant ActRIIBprotein above, wherein at least one alteration with respect to thesequence of SEQ ID NO:2 is positioned outside of the ligand bindingpocket. The variant ActRIIB protein above, wherein at least onealteration with respect to the sequence of SEQ ID NO:2 is a conservativealteration positioned within the ligand binding pocket. The variantActRIIB protein above, wherein at least one alteration with respect tothe sequence of SEQ ID NO:2 is an alteration at one or more positionsselected from the group consisting of K74, R40, Q53, K55, F82 and L79.The variant ActRIIB protein above, wherein the protein comprises atleast one N—X—S/T sequence at a position other than an endogenousN—X—S/T sequence of ActRIIB, and at a position outside of the ligandbinding pocket.

Other variant ActRIIB proteins are contemplated, such as the following.An ActRIIB protein comprising an amino acid sequence that is at least80% identical to the sequence of amino acids 29-109 of SEQ ID NO: 2, andwherein the protein comprises at least one N—X—S/T sequence at aposition other than an endogenous N—X—S/T sequence of ActRIIB, and at aposition outside of the ligand binding pocket. The variant ActRIIBprotein above, wherein the protein comprises an N at the positioncorresponding to position 24 of SEQ ID NO:2 and an S or T at theposition corresponding to position 26 of SEQ ID NO:2, and wherein thevariant ActRIIB protein inhibits signaling by activin, myostatin and/orGDF11 in a cell-based assay. The variant ActRIIB protein above, whereinthe protein comprises an R or K at the position corresponding toposition 64 of SEQ ID NO:2. The variant ActRIIB protein above, whereinat least one alteration with respect to the sequence of SEQ ID NO:2 is aconservative alteration positioned within the ligand binding pocket. Thevariant ActRIIB protein above, wherein at least one alteration withrespect to the sequence of SEQ ID NO:2 is an alteration at one or morepositions selected from the group consisting of K74, R40, Q53, K55, F82and L79. The variant ActRIIB protein above, wherein the protein is afusion protein further comprising a heterologous portion. Any of theabove variant ActRIIB fusion protein may be produced as a homodimer. Anyof the above ActRIIB fusion proteins may have a heterologous portionthat comprises a constant region from an IgG heavy chain, such as an Fcdomain.

In certain aspects, the disclosure provides nucleic acids encoding asoluble ActRIIB polypeptide, which do not encode a complete ActRIIBpolypeptide. An isolated polynucleotide may comprise a coding sequencefor a soluble ActRIIB polypeptide, such as described above. For example,an isolated nucleic acid may include a sequence coding for anextracellular domain (e.g., ligand-binding domain) of an ActRIIB and asequence that would code for part or all of the transmembrane domainand/or the cytoplasmic domain of an ActRIIB, but for a stop codonpositioned within the transmembrane domain or the cytoplasmic domain, orpositioned between the extracellular domain and the transmembrane domainor cytoplasmic domain. For example, an isolated polynucleotide maycomprise a full-length ActRIIB polynucleotide sequence such as SEQ IDNO: 4, or a partially truncated version, said isolated polynucleotidefurther comprising a transcription termination codon at least sixhundred nucleotides before the 3′-terminus or otherwise positioned suchthat translation of the polynucleotide gives rise to an extracellulardomain optionally fused to a truncated portion of a full-length ActRIIB.Nucleic acids disclosed herein may be operably linked to a promoter forexpression, and the disclosure provides cells transformed with suchrecombinant polynucleotides. Preferably the cell is a mammalian cellsuch as a CHO cell.

In certain aspects, the disclosure provides methods for making a solubleActRIIB polypeptide. Such a method may include expressing any of thenucleic acids (e.g., SEQ ID NO: 3) disclosed herein in a suitable cell,such as a Chinese hamster ovary (CHO) cell. Such a method may comprise:a) culturing a cell under conditions suitable for expression of thesoluble ActRIIB polypeptide, wherein said cell is transformed with asoluble ActRIIB expression construct; and b) recovering the solubleActRIIB polypeptide so expressed. Soluble ActRIIB polypeptides may berecovered as crude, partially purified or highly purified fractionsusing any of the well known techniques for obtaining protein from cellcultures.

In certain aspects, a soluble ActRIIB polypeptide disclosed herein maybe used in a method for treating a subject having a disorder associatedwith muscle loss or insufficient muscle growth. Such disorders includemuscle atrophy, muscular dystrophy, amyotrophic lateral sclerosis (ALS),and a muscle wasting disorder (e.g., cachexia, anorexia, DMD syndrome,BMD syndrome, AIDS wasting syndrome, muscular dystrophies, neuromusculardiseases, motor neuron diseases, diseases of the neuromuscular junction,and inflammatory myopathies). A method may comprise administering to asubject in need thereof an effective amount of a soluble ActRIIBpolypeptide.

In certain aspects, a soluble ActRIIB polypeptide disclosed herein maybe used in a method for decreasing the body fat content or reducing therate of increase in body fat content, and for treating a disorderassociated with undesirable body weight gain, such as obesity,non-insulin dependent diabetes mellitus (NIDDM), cardiovascular disease,cancer, hypertension, osteoarthritis, stroke, respiratory problems, andgall bladder disease. These methods may comprise administering to asubject in need thereof an effective amount of a soluble ActRIIBpolypeptide.

In certain specific aspects, a soluble ActRIIB polypeptide disclosedherein may be used in a method for treating a disorder associated withabnormal activity of GDF8. Such disorders include metabolic disorderssuch as type 2 diabetes, impaired glucose tolerance, metabolic syndrome(e.g., syndrome X), and insulin resistance induced by trauma (e.g.,burns or nitrogen imbalance); adipose tissue disorders (e.g., obesity);muscular dystrophy (including Duchenne muscular dystrophy); amyotrophiclateral sclerosis (ALS); muscle atrophy; organ atrophy; frailty; carpaltunnel syndrome; congestive obstructive pulmonary disease; sarcopenia,cachexia and other muscle wasting syndromes; osteoporosis;glucocorticoid-induced osteoporosis; osteopenia; osteoarthritis;osteoporosis-related fractures; low bone mass due to chronicglucocorticoid therapy, premature gonadal failure, androgen suppression,vitamin D deficiency, secondary hyperparathyroidism, nutritionaldeficiencies, and anorexia nervosa. The method may compriseadministering to a subject in need thereof an effective amount of asoluble ActRIIB polypeptide.

In certain aspects, the disclosure provides a method for identifying anagent that stimulates growth of a tissue such as bone, cartilage, muscleand fat. The method comprises: a) identifying a test agent that binds toa ligand-binding domain of an ActRIIB polypeptide competitively with asoluble ActRIIB polypeptide; and b) evaluating the effect of the agenton growth of the tissue.

In certain aspects, the disclosure provides methods for antagonizingactivity of an ActRIIB polypeptide or an ActRIIB ligand (e.g., GDF8,GDF11, activin, BMP7, and Nodal) in a cell. The methods comprisecontacting the cell with a soluble ActRIIB polypeptide. Optionally, theactivity of the ActRIIB polypeptide or the ActRIIB ligand is monitoredby a signaling transduction mediated by the ActRIIB/ActRIIB ligandcomplex, for example, by monitoring cell proliferation. The cells of themethods include an osteoblast, a chondrocyte, a myocyte, an adipocyteand a muscle cell.

In certain aspects, the disclosure provides uses of a soluble ActRIIBpolypeptide for making a medicament for the treatment of a disorder orcondition as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a human ActRIIB soluble (extracellular) polypeptidesequence (SEQ ID NO: 1). The C-terminal “tail” is underlined.

FIG. 2 shows human ActRIIB precursor protein sequence (SEQ ID NO: 2).The signal peptide is underlined; the extracellular domain is in bold(also referred to as SEQ ID NO: 1); and the potential N-linkedglycosylation sites are boxed.

FIG. 3 shows a nucleic acid sequence encoding a human ActRIIB soluble(extracellular) polypeptide, designed as SEQ ID NO: 3.

FIG. 4 shows a nucleic acid sequence encoding human ActRIIB precursorprotein, designed as SEQ ID NO: 4.

FIG. 5 shows body weight increases for mice treated with vehicle(diamonds), ActRIIB (R64 20-134)-mFc (squares) or the long half-lifeform, ActRIIB (R64 A24N 20-134)(triangles).

FIG. 6 shows weights of dissected muscles at the end of the study.Vehicle: left column (light shading) of each grouping; ActRIIB (R6420-134)-mFc: middle column (medium shading) of each grouping; ActRIIB(R64 A24N 20-134): right column (dark shading) of each grouping.

FIG. 7 shows grip strength measurements for PBS and murine ActRIIB (R64K74A 20-134)-mFc (or “K74A+15 tail”) treated SOD mice (white and blackbars, respectively). The figure illustrates the increased strength ofthe murine ActRIIB (R64 K74A 20-134)-mFc group compared to the PBS groupduring both the early (day 117) and the later (day 149) stages ofdisease. *P<0.05, two-tailed Student's t-test.

FIG. 8 shows the Kaplan-Meier survival comparison of PBS and ActRIIB(R64 K74A 20-134)-mFc treated SOD mice (white and black lines,respectively). The ActRIIB (R64 K74A 20-134)-mFc-treated cohort hasincreased the average number of days of survival compared to the PBSgroup.

FIG. 9 shows the percentage of body composition change in PBS andActRIIB (R64 20-134)-mFc HFD-fed mice (white and black bars,respectively). Treatment with murine ActRIIB (R64 20-134)-Fc proteinsignificantly reduces fat mass and increases lean tissue.

FIG. 10A shows muscle cross-sections of femoris muscle (4×magnification) from old mice. FIG. 10B shows muscle cross-sections offemoris muscle (4× magnification) from old mice treated with ActRIIB(R64 20-134)-mFc.

FIG. 11 shows the mean bodyweights for mice in a cancer cachexiaexperiment using CT26 colon cancer cells. Diamonds: untumoured, salinetreated animals; squares: untumoured, ActRIIB (R64 20-134)-mFc treatedmice; triangles: tumored, saline treated animals; “x”: tumored, ActRIIB(R64 20-134)-mFc treated mice (10 mg/kg); “*”: tumored, ActRIIB (R6420-134)-mFc treated mice (30 mg/kg); circle: tumored, ActRIIB (R6420-134)-mFc treated mice (10 mg/kg), treatment initiated at the time oftumor implant for a preventative modality.

FIG. 12 shows an alignment of human ActRIIA and ActRIIB with theresidues that are deduced herein, based on composite analysis ofmultiple ActRIIB and ActRIIA crystal structures to directly contactligand (the ligand binding pocket) indicated with boxes.

FIG. 13 shows a multiple sequence alignment of various vertebrateActRIIB proteins and human ActRIIA.

DETAILED DESCRIPTION 1. Overview

In certain aspects, the present invention relates to ActRIIBpolypeptides. As used herein, the term “ActRIIB” refers to a family ofactivin receptor type IIB (ActRIIB) proteins and ActRIIB-relatedproteins, derived from any species. Members of the ActRIIB family aregenerally all transmembrane proteins, composed of a ligand-bindingextracellular domain with cysteine-rich region, a transmembrane domain,and a cytoplasmic domain with predicted serine/threonine kinasespecificity. Amino acid sequences of human ActRIIA precursor protein(provided for comparison) and ActRIIB precursor protein are illustratedin FIG. 1 (SEQ ID NO: 1) and FIG. 2 (SEQ ID NO: 2), respectively.

The term “ActRIIB polypeptide” is used to refer to polypeptidescomprising any naturally occurring polypeptide of an ActRIIB familymember as well as any variants thereof (including mutants, fragments,fusions, and peptidomimetic forms) that retain a useful activity. Forexample, ActRIIB polypeptides include polypeptides derived from thesequence of any known ActRIIB having a sequence at least about 80%identical to the sequence of an ActRIIB polypeptide, and preferably atleast 85%, 90%, 95%, 97%, 99% or greater identity.

In a specific embodiment, the invention relates to soluble ActRIIBpolypeptides. As described herein, the term “soluble ActRIIBpolypeptide” generally refers to polypeptides comprising anextracellular domain of an ActRIIB protein. The term “soluble ActRIIBpolypeptide,” as used herein, includes any naturally occurringextracellular domain of an ActRIIB protein as well as any variantsthereof (including mutants, fragments and peptidomimetic forms) thatretain a useful activity. For example, the extracellular domain of anActRIIB protein binds to a ligand and is generally soluble. Examples ofsoluble ActRIIB polypeptides include ActRIIB soluble polypeptidesillustrated in FIG. 1 (SEQ ID NO: 1. Other examples of soluble ActRIIBpolypeptides comprise a signal sequence in addition to the extracellulardomain of an ActRIIB protein, see Example 1. The signal sequence can bea native signal sequence of an ActRIIB, or a signal sequence fromanother protein, such as a tissue plasminogen activator (TPA) signalsequence or a honey bee melatin (HBM) signal sequence.

TGF-β signals are mediated by heteromeric complexes of type I and typeII serine/threonine kinase receptors, which phosphorylate and activatedownstream Smad proteins upon ligand stimulation (Massague, 2000, Nat.Rev. Mol. Cell Biol. 1:169-178). These type I and type II receptors areall transmembrane proteins, composed of a ligand-binding extracellulardomain with cysteine-rich region, a transmembrane domain, and acytoplasmic domain with predicted serine/threonine specificity. Type Ireceptors are essential for signaling; and type II receptors arerequired for binding ligands and for expression of type I receptors.Type I and II activin receptors form a stable complex after ligandbinding, resulting in phosphorylation of type I receptors by type IIreceptors.

Two related type II receptors, ActRIIA and ActRIIB, have been identifiedas the type II receptors for activins (Mathews and Vale, 1991, Cell65:973-982; Attisano et al., 1992, Cell 68: 97-108). Besides activins,ActRIIA and ActRIIB can biochemically interact with several other TGF-βfamily proteins, including BMP7, Nodal, GDF8, and GDF11 (Yamashita etal., 1995, J. Cell Biol. 130:217-226; Lee and McPherron, 2001, Proc.Natl. Acad. Sci. 98:9306-9311; Yeo and Whitman, 2001, Mol. Cell 7:949-957; Oh et al., 2002, Genes Dev. 16:2749-54). Applicants have foundthat soluble ActRIIA-Fc fusion proteins and ActRIIB-Fc fusion proteinshave substantially different effects in vivo, with ActRIIA-Fc havingprimary effects on bone and ActRIIB-Fc having primary effects onskeletal muscle.

In certain embodiments, the present invention relates to antagonizing aligand of ActRIIB receptors (also referred to as an ActRIIB ligand) witha subject ActRIIB polypeptide (e.g., a soluble ActRIIB polypeptide).Thus, compositions and methods of the present invention are useful fortreating disorders associated with abnormal activity of one or moreligands of ActRIIB receptors. Exemplary ligands of ActRIIB receptorsinclude some TGF-β family members, such as activin, Nodal, GDF8, GDF11,and BMP7.

Activins are dimeric polypeptide growth factors and belong to theTGF-beta superfamily. There are three activins (A, B, and AB) that arehomo/heterodimers of two closely related β subunits (β_(A)β_(A),β_(B)β_(B), and β_(A)β_(B)). In the TGF-beta superfamily, activins areunique and multifunctional factors that can stimulate hormone productionin ovarian and placental cells, support neuronal cell survival,influence cell-cycle progress positively or negatively depending on celltype, and induce mesodermal differentiation at least in amphibianembryos (DePaolo et al., 1991, Proc SocEp Biol Med. 198:500-512; Dysonet al., 1997, Curr Biol. 7:81-84; Woodruff, 1998, Biochem Pharmacol.55:953-963). Moreover, erythroid differentiation factor (EDF) isolatedfrom the stimulated human monocytic leukemic cells was found to beidentical to activin A (Murata et al., 1988, PNAS, 85:2434). It wassuggested that activin A acts as a natural regulator of erythropoiesisin the bone marrow. In several tissues, activin signaling is antagonizedby its related heterodimer, inhibin. For example, during the release offollicle-stimulating hormone (FSH) from the pituitary, activin promotesFSH secretion and synthesis, while inhibin prevents FSH secretion andsynthesis. Other proteins that may regulate activin bioactivity and/orbind to activin include follistatin (FS), follistatin-related protein(FSRP), α₂-macroglobulin, Cerberus, and endoglin, which are describedbelow.

Nodal proteins have functions in mesoderm and endoderm induction andformation, as well as subsequent organization of axial structures suchas heart and stomach in early embryogenesis. It has been demonstratedthat dorsal tissue in a developing vertebrate embryo contributespredominantly to the axial structures of the notochord and pre-chordalplate while it recruits surrounding cells to form non-axial embryonicstructures. Nodal appears to signal through both type I and type IIreceptors and intracellular effectors known as Smad proteins. Recentstudies support the idea that ActRIIA and ActRIIB serve as type IIreceptors for Nodal (Sakuma et al., Genes Cells. 2002, 7:401-12). It issuggested that Nodal ligands interact with their co-factors (e.g.,cripto) to activate activin type I and type II receptors, whichphosphorylate Smad2. Nodal proteins are implicated in many eventscritical to the early vertebrate embryo, including mesoderm formation,anterior patterning, and left-right axis specification. Experimentalevidence has demonstrated that Nodal signaling activates pAR3-Lux, aluciferase reporter previously shown to respond specifically to activinand TGF-beta. However, Nodal is unable to induce pTlx2-Lux, a reporterspecifically responsive to bone morphogenetic proteins. Recent resultsprovide direct biochemical evidence that Nodal signaling is mediated byboth activin-TGF-beta pathway Smads, Smad2 and Smad3. Further evidencehas shown that the extracellular cripto protein is required for Nodalsignaling, making it distinct from activin or TGF-beta signaling.

Growth and Differentiation Factor-8 (GDF8) is also known as myostatin.GDF8 is a negative regulator of skeletal muscle mass. GDF8 is highlyexpressed in the developing and adult skeletal muscle. The GDF8 nullmutation in transgenic mice is characterized by a marked hypertrophy andhyperplasia of the skeletal muscle (McPherron et al., Nature, 1997,387:83-90). Similar increases in skeletal muscle mass are evident innaturally occurring mutations of GDF8 in cattle (Ashmore et al., 1974,Growth, 38:501-507; Swatland and Kieffer, J. Anim. Sci., 1994,38:752-757; McPherron and Lee, Proc. Natl. Acad. Sci. USA, 1997,94:12457-12461; and Kambadur et al., Genome Res., 1997, 7:910-915) and,strikingly, in humans (Schuelke et al., N Engl J Med 2004; 350:2682-8).Studies have also shown that muscle wasting associated withHIV-infection in humans is accompanied by increases in GDF8 proteinexpression (Gonzalez-Cadavid et al., PNAS, 1998, 95:14938-43). Inaddition, GDF8 can modulate the production of muscle-specific enzymes(e.g., creatine kinase) and modulate myoblast cell proliferation (WO00/43781). The GDF8 propeptide can noncovalently bind to the mature GDF8domain dimer, inactivating its biological activity (Miyazono et al.(1988) J. Biol. Chem., 263: 6407-6415; Wakefield et al. (1988) J. Biol.Chem., 263; 7646-7654; and Brown et al. (1990) Growth Factors, 3:35-43). Other proteins which bind to GDF8 or structurally relatedproteins and inhibit their biological activity include follistatin, andpotentially, follistatin-related proteins (Gamer et al. (1999) Dev.Biol., 208: 222-232).

Growth and Differentiation Factor-11 (GDF11), also known as BMP11, is asecreted protein (McPherron et al., 1999, Nat. Genet. 22: 260-264).GDF11 is expressed in the tail bud, limb bud, maxillary and mandibulararches, and dorsal root ganglia during mouse development (Nakashima etal., 1999, Mech. Dev. 80: 185-189). GDF11 plays a unique role inpatterning both mesodermal and neural tissues (Gamer et al., 1999, DevBiol., 208:222-32). GDF11 was shown to be a negative regulator ofchondrogenesis and myogenesis in developing chick limb (Gamer et al.,2001, Dev Biol. 229:407-20). The expression of GDF11 in muscle alsosuggests its role in regulating muscle growth in a similar way to GDF8.In addition, the expression of GDF11 in brain suggests that GDF11 mayalso possess activities that relate to the function of the nervoussystem. Interestingly, GDF11 was found to inhibit neurogenesis in theolfactory epithelium (Wu et al., 2003, Neuron. 37:197-207). Hence, GDF11may have in vitro and in vivo applications in the treatment of diseasessuch as muscle diseases and neurodegenerative diseases (e.g.,amyotrophic lateral sclerosis).

Bone morphogenetic protein (BMP7), also called osteogenic protein-1(OP-1), is well known to induce cartilage and bone formation. Inaddition, BMP7 regulates a wide array of physiological processes. Forexample, BMP7 may be the osteoinductive factor responsible for thephenomenon of epithelial osteogenesis. It is also found that BMP7 playsa role in calcium regulation and bone homeostasis. Like activin, BMP7binds to type II receptors, ActRIIA and IIB. However, BMP7 and activinrecruit distinct type I receptors into heteromeric receptor complexes.The major BMP7 type I receptor observed was ALK2, while activin boundexclusively to ALK4 (ActRIIB). BMP7 and activin elicited distinctbiological responses and activated different Smad pathways (Macias-Silvaet al., 1998, J Biol Chem. 273:25628-36).

In certain aspects, the present invention relates to the use of certainActRIIB polypeptides (e.g., soluble ActRIIB polypeptides) to antagonizethe signaling of ActRIIB ligands generally, in any process associatedwith ActRIIB activity. Optionally, ActRIIB polypeptides of the inventionmay antagonize one or more ligands of ActRIIB receptors, such asactivin, Nodal, GDF8, GDF11, and BMP7, and may therefore be useful inthe treatment of additional disorders.

Therefore, the present invention contemplates using ActRIIB polypeptidesin treating or preventing diseases or conditions that are associatedwith abnormal activity of an ActRIIB or an ActRIIB ligand. ActRIIB orActRIIB ligands are involved in the regulation of many criticalbiological processes. Due to their key functions in these processes,they may be desirable targets for therapeutic intervention. For example,ActRIIB polypeptides (e.g., soluble ActRIIB polypeptides) may be used totreat human or animal disorders or conditions. Example of such disordersor conditions include, but are not limited to, metabolic disorders suchas type 2 diabetes, impaired glucose tolerance, metabolic syndrome(e.g., syndrome X), and insulin resistance induced by trauma (e.g.,burns or nitrogen imbalance); adipose tissue disorders (e.g., obesity);muscle and neuromuscular disorders such as muscular dystrophy (includingDuchenne muscular dystrophy); amyotrophic lateral sclerosis (ALS);muscle atrophy; organ atrophy; frailty; carpal tunnel syndrome;congestive obstructive pulmonary disease; and sarcopenia, cachexia andother muscle wasting syndromes. Other examples include osteoporosis,especially in the elderly and/or postmenopausal women;glucocorticoid-induced osteoporosis; osteopenia; osteoarthritis; andosteoporosis-related fractures. Yet further examples include low bonemass due to chronic glucocorticoid therapy, premature gonadal failure,androgen suppression, vitamin D deficiency, secondaryhyperparathyroidism, nutritional deficiencies, and anorexia nervosa.These disorders and conditions are discussed below under “ExemplaryTherapeutic Uses.”

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this invention and in thespecific context where each term is used. Certain terms are discussedbelow or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the compositions and methods of theinvention and how to make and use them. The scope or meaning of any useof a term will be apparent from the specific context in which the termis used.

“About” and “approximately” shall generally mean an acceptable degree oferror for the quantity measured given the nature or precision of themeasurements. Typically, exemplary degrees of error are within 20percent (%), preferably within 10%, and more preferably within 5% of agiven value or range of values.

Alternatively, and particularly in biological systems, the terms “about”and “approximately” may mean values that are within an order ofmagnitude, preferably within 5-fold and more preferably within 2-fold ofa given value. Numerical quantities given herein are approximate unlessstated otherwise, meaning that the term “about” or “approximately” canbe inferred when not expressly stated.

The methods of the invention may include steps of comparing sequences toeach other, including wild-type sequence to one or more mutants(sequence variants). Such comparisons typically comprise alignments ofpolymer sequences, e.g., using sequence alignment programs and/oralgorithms that are well known in the art (for example, BLAST, FASTA andMEGALIGN, to name a few). The skilled artisan can readily appreciatethat, in such alignments, where a mutation contains a residue insertionor deletion, the sequence alignment will introduce a “gap” (typicallyrepresented by a dash, or “A”) in the polymer sequence not containingthe inserted or deleted residue.

“Homologous,” in all its grammatical forms and spelling variations,refers to the relationship between two proteins that possess a “commonevolutionary origin,” including proteins from superfamilies in the samespecies of organism, as well as homologous proteins from differentspecies of organism. Such proteins (and their encoding nucleic acids)have sequence homology, as reflected by their sequence similarity,whether in terms of percent identity or by the presence of specificresidues or motifs and conserved positions.

The term “sequence similarity,” in all its grammatical forms, refers tothe degree of identity or correspondence between nucleic acid or aminoacid sequences that may or may not share a common evolutionary origin.

However, in common usage and in the instant application, the term“homologous,” when modified with an adverb such as “highly,” may referto sequence similarity and may or may not relate to a commonevolutionary origin.

2. ActRIIB Polypeptides

In certain aspects, the invention relates to ActRIIB variantpolypeptides (e.g., soluble ActRIIB polypeptides). Optionally, thefragments, functional variants, and modified forms have similar or thesame biological activities of their corresponding wild-type ActRIIBpolypeptides. For example, an ActRIIB variant of the invention may bindto and inhibit function of an ActRIIB ligand (e.g., activin A, activinAB, activin B, Nodal, GDF8, GDF11 or BMP7). Optionally, an ActRIIBpolypeptide modulates growth of tissues such as bone, cartilage, muscleor fat. Examples of ActRIIB polypeptides include human ActRIIB precursorpolypeptide (SEQ ID NO: 2), and soluble human ActRIIB polypeptides(e.g., SEQ ID NOs: 1, 5, 6 and 12).

The disclosure identifies functionally active portions and variants ofActRIIB. Applicants have ascertained that an Fc fusion protein havingthe sequence disclosed by Hilden et al. (Blood. 1994 Apr. 15;83(8):2163-70), which has an Alanine at the position corresponding toamino acid 64 of SEQ ID NO: 2 (A64), has a relatively low affinity foractivin and GDF-11. By contrast, the same Fc fusion protein with anArginine at position 64 (R64) has an affinity for activin and GDF-11 inthe low nanomolar to high picomolar range. Therefore, a sequence with anR64 is used as the wild-type reference sequence for human ActRIIB inthis disclosure.

Attisano et al. (Cell. 1992 Jan. 10; 68(1):97-108) showed that adeletion of the proline knot at the C-terminus of the extracellulardomain of ActRIIB reduced the affinity of the receptor for activin. Datapresented here shows that an ActRIIB-Fc fusion protein containing aminoacids 20-119 of SEQ ID NO:2, “ActRIIB (20-119)-Fc” has reduced bindingto GDF-11 and activin relative to an ActRIIB (20-134)-Fc, which includesthe proline knot region and the complete juxtamembrane domain. However,an ActRIIB (20-129)-Fc protein retains similar but somewhat reducedactivity relative to the wild type, even though the proline knot regionis disrupted. Thus, ActRIIB extracellular domains that stop at aminoacid 134, 133, 132, 131, 130 and 129 are all expected to be active, butconstructs stopping at 134 or 133 may be most active. Similarly,mutations at any of residues 129-134 are not expected to alter ligandbinding affinity by large margins. In support of this, mutations of P129and P130 do not substantially decrease ligand binding. Therefore, anActRIIB-Fc fusion protein may end as early as amino acid 109 (the finalcysteine), however, forms ending at or between 109 and 119 are expectedto have reduced ligand binding. Amino acid 119 is poorly conserved andso is readily altered or truncated. Forms ending at 128 or later retainligand binding activity. Forms ending at or between 119 and 127 willhave an intermediate binding ability. Any of these forms may bedesirable to use, depending on the clinical or experimental setting.

At the N-terminus of ActRIIB, it is expected that a protein beginning atamino acid 29 or before will retain ligand binding activity. Amino acid29 represents the initial cysteine. An alanine to asparagine mutation atposition 24 introduces an N-linked glycosylation sequence withoutsubstantially affecting ligand binding. This confirms that mutations inthe region between the signal cleavage peptide and the cysteinecross-linked region, corresponding to amino acids 20-29 are welltolerated. In particular, constructs beginning at position 20, 21, 22,23 and 24 will retain activity, and constructs beginning at positions25, 26, 27, 28 and 29 are also expected to retain activity. Data shownin the Examples demonstrates that, surprisingly, a construct beginningat 22, 23, 24 or 25 will have the most activity.

Taken together, an active portion of ActRIIB comprises amino acids29-109 of SEQ ID NO:2, and constructs may, for example, begin at aresidue corresponding to amino acids 20-29 and end at a positioncorresponding to amino acids 109-134. Other examples include constructsthat begin at a position from 20-29 or 21-29 and end at a position from119-134, 119-133 or 129-134, 129-133. Other examples include constructsthat begin at a position from 20-24 (or 21-24, or 22-25) and end at aposition from 109-134 (or 109-133), 119-134 (or 119-133) or 129-134 (or129-133). Variants within these ranges are also contemplated,particularly those having at least 80%, 85%, 90%, 95% or 99% identity tothe corresponding portion of SEQ ID NO:4.

The disclosure includes the results of an analysis of composite ActRIIBstructures, shown in FIG. 22, demonstrating that the ligand bindingpocket is defined by residues Y31, N33, N35, L38 through T41, E47, E50,Q53 through K55, L57, H58, Y60, S62, K74, W78 through N83, Y85, R87,A92, and E94 through F101. At these positions, it is expected thatconservative mutations will be tolerated, although a K74A mutation iswell-tolerated, as are R40A, K55A, F82A and mutations at position L79.R40 is a K in Xenopus, indicating that basic amino acids at thisposition will be tolerated. Q53 is R in bovine ActRIIB and K in XenopusActRIIB, and therefore amino acids including R, K, Q, N and H will betolerated at this position. Thus, a general formula for an activeActRIIB variant protein is one that comprises amino acids 29-109, butoptionally beginning at a position ranging from 20-24 or 22-25 andending at a position ranging from 129-134, and comprising no more than1, 2, 5, 10 or 15 conservative amino acid changes in the ligand bindingpocket, and zero, one or more non-conservative alterations at positions40, 53, 55, 74, 79 and/or 82 in the ligand binding pocket. Such aprotein may retain greater than 80%, 90%, 95% or 99% sequence identityto the sequence of amino acids 29-109 of SEQ ID NO:4. Sites outside thebinding pocket, at which variability may be particularly well tolerated,include the amino and carboxy termini of the extracellular domain (asnoted above), and positions 42-46 and 65-73. An asparagine to alaninealteration at position 65 (N65A) actually improves ligand binding in theA64 background, and is thus expected to have no detrimental effect onligand binding in the R64 background. This change probably eliminatesglycosylation at N65 in the A64 background, thus demonstrating that asignificant change in this region is likely to be tolerated. While anR64A change is poorly tolerated, R64K is well-tolerated, and thusanother basic residue, such as H may be tolerated at position 64.

ActRIIB is well-conserved across nearly all vertebrates, with largestretches of the extracellular domain conserved completely. Many of theligands that bind to ActRIIB are also highly conserved. Accordingly,comparisons of ActRIIB sequences from various vertebrate organismsprovide insights into residues that may be altered. Therefore, anactive, human ActRIIB variant may include one or more amino acids atcorresponding positions from the sequence of another vertebrate ActRIIB,or may include a residue that is similar to that in the human or othervertebrate sequence. The following examples illustrate this approach todefining an active ActRIIB variant. L46 is a valine in Xenopus ActRIIB,and so this position may be altered, and optionally may be altered toanother hydrophobic residue, such as V, I or F, or a non-polar residuesuch as A. E52 is a K in Xenopus, indicating that this site may betolerant of a wide variety of changes, including polar residues, such asE, D, K, R, H, S, T, P, G, Y and probably A. T93 is a K in Xenopus,indicating that a wide structural variation is tolerated at thisposition, with polar residues favored, such as S, K, R, E, D, H, G, P, Gand Y. F108 is a Y in Xenopus, and therefore Y or other hydrophobicgroup, such as I, V or L should be tolerated. E111 is K in Xenopus,indicating that charged residues will be tolerated at this position,including D, R, K and H, as well as Q and N. R112 is K in Xenopus,indicating that basic residues are tolerated at this position, includingR and H. A at position 119 is relatively poorly conserved, and appearsas P in rodents and V in Xenopus, thus essentially any amino acid shouldbe tolerated at this position.

The disclosure demonstrates that the addition of a further N-linkedglycosylation site (N—X—S/T) increases the serum half-life of anActRIIB-Fc fusion protein, relative to the ActRIIB (R64)-Fc form. Byintroducing an asparagine at position 24 (A24N construct), an NXTsequence is created that confers a longer half-life. Other NX(T/S)sequences are found at 42-44 (NQS) and 65-67 (NSS), although the lattermay not be efficiently glycosylated with the R at position 64. N—X—S/Tsequences may be generally introduced at positions outside the ligandbinding pocket defined in FIG. 12. Particularly suitable sites for theintroduction of non-endogenous N—X—S/T sequences include amino acids20-29, 20-24, 22-25, 109-134, 120-134 or 129-134. N—X—S/T sequences mayalso be introduced into the linker between the ActRIIB sequence and theFc or other fusion component. Such a site may be introduced with minimaleffort by introducing an N in the correct position with respect to apre-existing S or T, or by introducing an S or T at a positioncorresponding to a pre-existing N. Thus, desirable alterations thatwould create an N-linked glycosylation site are: A24N, R64N, S67N(possibly combined with an N65A alteration), E106N, R112N, G120N, E123N,P129N, A132N, R112S and R112T. Any S that is predicted to beglycosylated may be altered to a T without creating an immunogenic site,because of the protection afforded by the glycosylation. Likewise, any Tthat is predicted to be glycosylated may be altered to an S. Thus thealterations S67T and S44T are contemplated. Likewise, in an A24Nvariant, an S26T alteration may be used. Accordingly, an ActRIIB variantmay include one or more additional, non-endogenous N-linkedglycosylation consensus sequences.

Position L79 may be altered to confer altered activin—myostatin (GDF-11)binding properties. L79A or L79P reduces GDF-11 binding to a greaterextent than activin binding. L79E or L79D retains GDF-11 binding.Remarkably, the L79E and L79D variants have greatly reduced activinbinding. In vivo experiments indicate that these non-activin receptorsretain significant ability to increase muscle mass but show decreasedeffects on other tissues. These data demonstrate the desirability andfeasibility for obtaining polypeptides with reduced effects on activin.

The variations described may be combined in various ways. Additionally,the results of mutagenesis program described herein indicate that thereare amino acid positions in ActRIIb that are often beneficial toconserve. These include position 64 (basic amino acid), position 80(acidic or hydrophobic amino acid), position 78 (hydrophobic, andparticularly tryptophan), position 37 (acidic, and particularly asparticor glutamic acid), position 56 (basic amino acid), position 60(hydrophobic amino acid, particularly phenylalanine or tyrosine). Thus,in each of the variants disclosed herein, the disclosure provides aframework of amino acids that may be conserved. Other positions that maybe desirable to conserve are as follows: position 52 (acidic aminoacid), position 55 (basic amino acid), position 81 (acidic), 98 (polaror charged, particularly E, D, R or K).

In certain embodiments, isolated fragments of the ActRIIB polypeptidescan be obtained by screening polypeptides recombinantly produced fromthe corresponding fragment of the nucleic acid encoding an ActRIIBpolypeptide (e.g., SEQ ID NOs: 3 and 4). In addition, fragments can bechemically synthesized using techniques known in the art such asconventional Merrifield solid phase f-Moc or t-Boc chemistry. Thefragments can be produced (recombinantly or by chemical synthesis) andtested to identify those peptidyl fragments that can function, forexample, as antagonists (inhibitors) or agonists (activators) of anActRIIB protein or an ActRIIB ligand.

In certain embodiments, a functional variant of the ActRIIB polypeptideshas an amino acid sequence that is at least 75% identical to an aminoacid sequence selected from SEQ ID NOs: 3, 4 and 10. In certain cases,the functional variant has an amino acid sequence at least 80%, 85%,90%, 95%, 97%, 98%, 99% or 100% identical to an amino acid sequenceselected from SEQ ID NOs: 3, 4 and 10.

In certain embodiments, the present invention contemplates makingfunctional variants by modifying the structure of an ActRIIB polypeptidefor such purposes as enhancing therapeutic efficacy, or stability (e.g.,ex vivo shelf life and resistance to proteolytic degradation in vivo).Modified ActRIIB polypeptides can also be produced, for instance, byamino acid substitution, deletion, or addition. For instance, it isreasonable to expect that an isolated replacement of a leucine with anisoleucine or valine, an aspartate with a glutamate, a threonine with aserine, or a similar replacement of an amino acid with a structurallyrelated amino acid (e.g., conservative mutations) will not have a majoreffect on the biological activity of the resulting molecule.Conservative replacements are those that take place within a family ofamino acids that are related in their side chains. Whether a change inthe amino acid sequence of an ActRIIB polypeptide results in afunctional homolog can be readily determined by assessing the ability ofthe variant ActRIIB polypeptide to produce a response in cells in afashion similar to the wild-type ActRIIB polypeptide, or to bind to oneor more ligands, such as activin, GDF-11 or myostatin in a fashionsimilar to wild type.

In certain specific embodiments, the present invention contemplatesmaking mutations in the extracellular domain (also referred to asligand-binding domain) of an ActRIIB polypeptide such that the variant(or mutant) ActRIIB polypeptide has altered ligand-binding activities(e.g., binding affinity or binding specificity). In certain cases, suchvariant ActRIIB polypeptides have altered (elevated or reduced) bindingaffinity for a specific ligand. In other cases, the variant ActRIIBpolypeptides have altered binding specificity for their ligands.

For example, the disclosure provides variant ActRIIB polypeptides thatpreferentially bind to GDF8/GDF11 relative to activins. The disclosurefurther establishes the desirability of such polypeptides for reducingoff-target effects, although such selective variants may be lessdesirable for the treatment of severe diseases where very large gains inmuscle mass may be needed for therapeutic effect and where some level ofoff-target effect is acceptable. For example, amino acid residues of theActRIIB protein, such as E39, K55, Y60, K74, W78, D80, and F101, are inthe ligand-binding pocket and mediate binding to its ligands such asactivin and GDF8. Thus, the present invention provides an alteredligand-binding domain (e.g., GDF8-binding domain) of an ActRIIBreceptor, which comprises one or more mutations at those amino acidresidues. Optionally, the altered ligand-binding domain can haveincreased selectivity for a ligand such as GDF8 relative to a wild-typeligand-binding domain of an ActRIIB receptor. To illustrate, thesemutations increase the selectivity of the altered ligand-binding domainfor GDF8 over activin. Optionally, the altered ligand-binding domain hasa ratio of K_(d) for activin binding to K_(d) for GDF8 binding that isat least 2, 5, 10, or even 100 fold greater relative to the ratio forthe wild-type ligand-binding domain. Optionally, the alteredligand-binding domain has a ratio of IC₅₀ for inhibiting activin to IC₅₀for inhibiting GDF8 that is at least 2, 5, 10, or even 100 fold greaterrelative to the wild-type ligand-binding domain. Optionally, the alteredligand-binding domain inhibits GDF8 with an IC₅₀ at least 2, 5, 10, oreven 100 times less than the IC₅₀ for inhibiting activin.

As a specific example, the positively-charged amino acid residue Asp(D80) of the ligand-binding domain of ActRIIB can be mutated to adifferent amino acid residue such that the variant ActRIIB polypeptidepreferentially binds to GDF8, but not activin. Preferably, the D80residue is changed to an amino acid residue selected from the groupconsisting of: a uncharged amino acid residue, a negative amino acidresidue, and a hydrophobic amino acid residue. As a further specificexample, the hydrophobic residue, L79, can be altered to the acidicamino acids aspartic acid or glutamic acid to greatly reduce activinbinding while retaining GDF11 binding. As will be recognized by one ofskill in the art, most of the described mutations, variants ormodifications may be made at the nucleic acid level or, in some cases,by post translational modification or chemical synthesis. Suchtechniques are well known in the art.

In certain embodiments, the present invention contemplates specificmutations of the ActRIIB polypeptides so as to alter the glycosylationof the polypeptide. Exemplary glycosylation sites in ActRIIBpolypeptides are illustrated in FIG. 2. Such mutations may be selectedso as to introduce or eliminate one or more glycosylation sites, such asO-linked or N-linked glycosylation sites. Asparagine-linkedglycosylation recognition sites generally comprise a tripeptidesequence, asparagine-X-threonine (where “X” is any amino acid) which isspecifically recognized by appropriate cellular glycosylation enzymes.The alteration may also be made by the addition of, or substitution by,one or more serine or threonine residues to the sequence of thewild-type ActRIIB polypeptide (for O-linked glycosylation sites). Avariety of amino acid substitutions or deletions at one or both of thefirst or third amino acid positions of a glycosylation recognition site(and/or amino acid deletion at the second position) results innon-glycosylation at the modified tripeptide sequence. Another means ofincreasing the number of carbohydrate moieties on an ActRIIB polypeptideis by chemical or enzymatic coupling of glycosides to the ActRIIBpolypeptide. Depending on the coupling mode used, the sugar(s) may beattached to (a) arginine and histidine; (b) free carboxyl groups; (c)free sulfhydryl groups such as those of cysteine; (d) free hydroxylgroups such as those of serine, threonine, or hydroxyproline; (e)aromatic residues such as those of phenylalanine, tyrosine, ortryptophan; or (f) the amide group of glutamine. These methods aredescribed in WO 87/05330 published Sep. 11, 1987, and in Aplin andWriston (1981) CRC Crit. Rev. Biochem., pp. 259-306, incorporated byreference herein. Removal of one or more carbohydrate moieties presenton an ActRIIB polypeptide may be accomplished chemically and/orenzymatically. Chemical deglycosylation may involve, for example,exposure of the ActRIIB polypeptide to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving the aminoacid sequence intact. Chemical deglycosylation is further described byHakimuddin et al. (1987) Arch. Biochem. Biophys. 259:52 and by Edge etal. (1981) Anal. Biochem. 118:131. Enzymatic cleavage of carbohydratemoieties on ActRIIB polypeptides can be achieved by the use of a varietyof endo- and exo-glycosidases as described by Thotakura et al. (1987)Meth. Enzymol. 138:350. The sequence of an ActRIIB polypeptide may beadjusted, as appropriate, depending on the type of expression systemused, as mammalian, yeast, insect and plant cells may all introducediffering glycosylation patterns that can be affected by the amino acidsequence of the peptide. In general, ActRIIB proteins for use in humanswill be expressed in a mammalian cell line that provides properglycosylation, such as HEK293 or CHO cell lines, although othermammalian expression cell lines are expected to be useful as well.

This disclosure further contemplates a method of generating variants,particularly sets of combinatorial variants of an ActRIIB polypeptide,including, optionally, truncation variants; pools of combinatorialmutants are especially useful for identifying functional variantsequences. The purpose of screening such combinatorial libraries may beto generate, for example, ActRIIB polypeptide variants which havealtered properties, such as altered pharmacokinetics, or altered ligandbinding. A variety of screening assays are provided below, and suchassays may be used to evaluate variants. For example, an ActRIIBpolypeptide variant may be screened for ability to bind to an ActRIIBpolypeptide, to prevent binding of an ActRIIB ligand to an ActRIIBpolypeptide.

The activity of an ActRIIB polypeptide or its variants may also betested in a cell-based or in vivo assay. For example, the effect of anActRIIB polypeptide variant on the expression of genes involved in boneproduction in an osteoblast or precursor may be assessed. This may, asneeded, be performed in the presence of one or more recombinant ActRIIBligand protein (e.g., BMP7), and cells may be transfected so as toproduce an ActRIIB polypeptide and/or variants thereof, and optionally,an ActRIIB ligand. Likewise, an ActRIIB polypeptide may be administeredto a mouse or other animal, and one or more bone properties, such asdensity or volume may be assessed. The healing rate for bone fracturesmay also be evaluated. Similarly, the activity of an ActRIIB polypeptideor its variants may be tested in muscle cells, adipocytes, and neuronalcells for any effect on growth of these cells, for example, by theassays as described below. Such assays are well known and routine in theart. A SMAD-responsive reporter gene may be used in such cell lines tomonitor effects on downstream signaling.

Combinatorially-derived variants can be generated which have a selectivepotency relative to a naturally occurring ActRIIB polypeptide. Suchvariant proteins, when expressed from recombinant DNA constructs, can beused in gene therapy protocols. Likewise, mutagenesis can give rise tovariants which have intracellular half-lives dramatically different thanthe corresponding a wild-type ActRIIB polypeptide. For example, thealtered protein can be rendered either more stable or less stable toproteolytic degradation or other processes which result in destructionof, or otherwise inactivation of a native ActRIIB polypeptide. Suchvariants, and the genes which encode them, can be utilized to alterActRIIB polypeptide levels by modulating the half-life of the ActRIIBpolypeptides. For instance, a short half-life can give rise to moretransient biological effects and, when part of an inducible expressionsystem, can allow tighter control of recombinant ActRIIB polypeptidelevels within the cell.

In certain embodiments, the ActRIIB polypeptides of the invention mayfurther comprise post-translational modifications in addition to anythat are naturally present in the ActRIIB polypeptides. Suchmodifications include, but are not limited to, acetylation,carboxylation, glycosylation, phosphorylation, lipidation, andacylation. As a result, the modified ActRIIB polypeptides may containnon-amino acid elements, such as polyethylene glycols, lipids, poly- ormono-saccharide, and phosphates. Effects of such non-amino acid elementson the functionality of a ActRIIB polypeptide may be tested as describedherein for other ActRIIB polypeptide variants. When an ActRIIBpolypeptide is produced in cells by cleaving a nascent form of theActRIIB polypeptide, post-translational processing may also be importantfor correct folding and/or function of the protein. Different cells(such as CHO, HeLa, MDCK, 293, WI38, NIH-3T3 or HEK293) have specificcellular machinery and characteristic mechanisms for suchpost-translational activities and may be chosen to ensure the correctmodification and processing of the ActRIIB polypeptides.

In certain aspects, functional variants or modified forms of the ActRIIBpolypeptides include fusion proteins having at least a portion of theActRIIB polypeptides and one or more fusion domains. Well known examplesof such fusion domains include, but are not limited to, polyhistidine,Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A,protein G, an immunoglobulin heavy chain constant region (e.g., an Fc),maltose binding protein (MBP), or human serum albumin. A fusion domainmay be selected so as to confer a desired property. For example, somefusion domains are particularly useful for isolation of the fusionproteins by affinity chromatography. For the purpose of affinitypurification, relevant matrices for affinity chromatography, such asglutathione-, amylase-, and nickel- or cobalt-conjugated resins areused. Many of such matrices are available in “kit” form, such as thePharmacia GST purification system and the QIAexpression™ system (Qiagen)useful with (HIS₆) fusion partners. As another example, a fusion domainmay be selected so as to facilitate detection of the ActRIIBpolypeptides. Examples of such detection domains include the variousfluorescent proteins (e.g., GFP) as well as “epitope tags,” which areusually short peptide sequences for which a specific antibody isavailable. Well known epitope tags for which specific monoclonalantibodies are readily available include FLAG, influenza virushaemagglutinin (HA), and c-myc tags. In some cases, the fusion domainshave a protease cleavage site, such as for Factor Xa or Thrombin, whichallows the relevant protease to partially digest the fusion proteins andthereby liberate the recombinant proteins therefrom. The liberatedproteins can then be isolated from the fusion domain by subsequentchromatographic separation. In certain preferred embodiments, an ActRIIBpolypeptide is fused with a domain that stabilizes the ActRIIBpolypeptide in vivo (a “stabilizer” domain). By “stabilizing” is meantanything that increases serum half life, regardless of whether this isbecause of decreased destruction, decreased clearance by the kidney, orother pharmacokinetic effect. Fusions with the Fc portion of animmunoglobulin are known to confer desirable pharmacokinetic propertieson a wide range of proteins. Likewise, fusions to human serum albumincan confer desirable properties. Other types of fusion domains that maybe selected include multimerizing (e.g., dimerizing, tetramerizing)domains and functional domains (that confer an additional biologicalfunction, such as further stimulation of muscle growth).

As a specific example, the present invention provides a fusion proteinas a GDF8 antagonist which comprises an extracellular (e.g.,GDF8-binding) domain fused to an Fc domain (e.g., SEQ ID NO: 13).

THTCPPCPAPELLGGPSVFLFPPKPKDILMISRTPEVTCVVVD(A)VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK(A)VSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGPFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN(A)HYTQKSLSLSPGK*

Preferably, the Fc domain has one or more mutations at residues such asAsp-265, lysine 322, and Asn-434. In certain cases, the mutant Fc domainhaving one or more of these mutations (e.g., Asp-265 mutation) hasreduced ability of binding to the Fcγ receptor relative to a wildtype Fcdomain. In other cases, the mutant Fc domain having one or more of thesemutations (e.g., Asn-434 mutation) has increased ability of binding tothe MHC class I-related Fc-receptor (FcRN) relative to a wildtype Fcdomain.

It is understood that different elements of the fusion proteins may bearranged in any manner that is consistent with the desiredfunctionality. For example, an ActRIIB polypeptide may be placedC-terminal to a heterologous domain, or, alternatively, a heterologousdomain may be placed C-terminal to an ActRIIB polypeptide. The ActRIIBpolypeptide domain and the heterologous domain need not be adjacent in afusion protein, and additional domains or amino acid sequences may beincluded C- or N-terminal to either domain or between the domains.

In certain embodiments, the ActRIIB polypeptides of the presentinvention contain one or more modifications that are capable ofstabilizing the ActRIIB polypeptides. For example, such modificationsenhance the in vitro half life of the ActRIIB polypeptides, enhancecirculatory half life of the ActRIIB polypeptides or reducingproteolytic degradation of the ActRIIB polypeptides. Such stabilizingmodifications include, but are not limited to, fusion proteins(including, for example, fusion proteins comprising an ActRIIBpolypeptide and a stabilizer domain), modifications of a glycosylationsite (including, for example, addition of a glycosylation site to anActRIIB polypeptide), and modifications of carbohydrate moiety(including, for example, removal of carbohydrate moieties from anActRIIB polypeptide). In the case of fusion proteins, an ActRIIBpolypeptide is fused to a stabilizer domain such as an IgG molecule(e.g., an Fc domain). As used herein, the term “stabilizer domain” notonly refers to a fusion domain (e.g., Fc) as in the case of fusionproteins, but also includes nonproteinaceous modifications such as acarbohydrate moiety, or nonproteinaceous polymer, such as polyethyleneglycol.

In certain embodiments, the present invention makes available isolatedand/or purified forms of the ActRIIB polypeptides, which are isolatedfrom, or otherwise substantially free of, other proteins.

In certain embodiments, ActRIIB polypeptides (unmodified or modified) ofthe invention can be produced by a variety of art-known techniques. Forexample, such ActRIIB polypeptides can be synthesized using standardprotein chemistry techniques such as those described in Bodansky, M.Principles of Peptide Synthesis, Springer Verlag, Berlin (1993) andGrant G. A. (ed.), Synthetic Peptides: A User's Guide, W. H. Freeman andCompany, New York (1992). In addition, automated peptide synthesizersare commercially available (e.g., Advanced ChemTech Model 396;Milligen/Biosearch 9600). Alternatively, the ActRIIB polypeptides,fragments or variants thereof may be recombinantly produced usingvarious expression systems (e.g., E. coli, Chinese Hamster Ovary cells,COS cells, baculovirus) as is well known in the art (also see below). Ina further embodiment, the modified or unmodified ActRIIB polypeptidesmay be produced by digestion of naturally occurring or recombinantlyproduced full-length ActRIIB polypeptides by using, for example, aprotease, e.g., trypsin, thermolysin, chymotrypsin, pepsin, or pairedbasic amino acid converting enzyme (PACE). Computer analysis (using acommercially available software, e.g., MacVector, Omega, PCGene,Molecular Simulation, Inc.) can be used to identify proteolytic cleavagesites. Alternatively, such ActRIIB polypeptides may be produced fromnaturally occurring or recombinantly produced full-length ActRIIBpolypeptides such as standard techniques known in the art, such as bychemical cleavage (e.g., cyanogen bromide, hydroxylamine).

3. Nucleic Acids Encoding ActRIIB Polypeptides

In certain aspects, the invention provides isolated and/or recombinantnucleic acids encoding any of the ActRIIB polypeptides (e.g., solubleActRIIB polypeptides), including any of the variants disclosed herein.For example, SEQ ID NO: 4 encodes a naturally occurring ActRIIBprecursor polypeptide, while SEQ ID NO: 3 encodes a soluble ActRIIBpolypeptide. The subject nucleic acids may be single-stranded or doublestranded. Such nucleic acids may be DNA or RNA molecules. These nucleicacids are may be used, for example, in methods for making ActRIIBpolypeptides or as direct therapeutic agents (e.g., in a gene therapyapproach).

In certain aspects, the subject nucleic acids encoding ActRIIBpolypeptides are further understood to include nucleic acids that arevariants of SEQ ID NO: 3. Variant nucleotide sequences include sequencesthat differ by one or more nucleotide substitutions, additions ordeletions, such as allelic variants; and will, therefore, include codingsequences that differ from the nucleotide sequence of the codingsequence designated in SEQ ID NO: 4.

In certain embodiments, the invention provides isolated or recombinantnucleic acid sequences that are at least 80%, 85%, 90%, 95%, 97%, 98%,99% or 100% identical to SEQ ID NO: 3. One of ordinary skill in the artwill appreciate that nucleic acid sequences complementary to SEQ ID NO:3, and variants of SEQ ID NO: 3 are also within the scope of thisinvention. In further embodiments, the nucleic acid sequences of theinvention can be isolated, recombinant, and/or fused with a heterologousnucleotide sequence, or in a DNA library.

In other embodiments, nucleic acids of the invention also includenucleotide sequences that hybridize under highly stringent conditions tothe nucleotide sequence designated in SEQ ID NO: 3, complement sequenceof SEQ ID NO: 3, or fragments thereof. As discussed above, one ofordinary skill in the art will understand readily that appropriatestringency conditions which promote DNA hybridization can be varied. Oneof ordinary skill in the art will understand readily that appropriatestringency conditions which promote DNA hybridization can be varied. Forexample, one could perform the hybridization at 6.0× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by a wash of2.0×SSC at 50° C. For example, the salt concentration in the wash stepcan be selected from a low stringency of about 2.0×SSC at 50° C. to ahigh stringency of about 0.2×SSC at 50° C. In addition, the temperaturein the wash step can be increased from low stringency conditions at roomtemperature, about 22° C., to high stringency conditions at about 65° C.Both temperature and salt may be varied, or temperature or saltconcentration may be held constant while the other variable is changed.In one embodiment, the invention provides nucleic acids which hybridizeunder low stringency conditions of 6×SSC at room temperature followed bya wash at 2×SSC at room temperature.

Isolated nucleic acids which differ from the nucleic acids as set forthin SEQ ID NO: 3 due to degeneracy in the genetic code are also withinthe scope of the invention. For example, a number of amino acids aredesignated by more than one triplet. Codons that specify the same aminoacid, or synonyms (for example, CAU and CAC are synonyms for histidine)may result in “silent” mutations which do not affect the amino acidsequence of the protein. However, it is expected that DNA sequencepolymorphisms that do lead to changes in the amino acid sequences of thesubject proteins will exist among mammalian cells. One skilled in theart will appreciate that these variations in one or more nucleotides (upto about 3-5% of the nucleotides) of the nucleic acids encoding aparticular protein may exist among individuals of a given species due tonatural allelic variation. Any and all such nucleotide variations andresulting amino acid polymorphisms are within the scope of thisinvention.

In certain embodiments, the recombinant nucleic acids of the inventionmay be operably linked to one or more regulatory nucleotide sequences inan expression construct. Regulatory nucleotide sequences will generallybe appropriate to the host cell used for expression. Numerous types ofappropriate expression vectors and suitable regulatory sequences areknown in the art for a variety of host cells. Typically, said one ormore regulatory nucleotide sequences may include, but are not limitedto, promoter sequences, leader or signal sequences, ribosomal bindingsites, transcriptional start and termination sequences, translationalstart and termination sequences, and enhancer or activator sequences.Constitutive or inducible promoters as known in the art are contemplatedby the invention. The promoters may be either naturally occurringpromoters, or hybrid promoters that combine elements of more than onepromoter. An expression construct may be present in a cell on anepisome, such as a plasmid, or the expression construct may be insertedin a chromosome. In a preferred embodiment, the expression vectorcontains a selectable marker gene to allow the selection of transformedhost cells. Selectable marker genes are well known in the art and willvary with the host cell used.

In certain aspects of the invention, the subject nucleic acid isprovided in an expression vector comprising a nucleotide sequenceencoding an ActRIIB polypeptide and operably linked to at least oneregulatory sequence. Regulatory sequences are art-recognized and areselected to direct expression of the ActRIIB polypeptide. Accordingly,the term regulatory sequence includes promoters, enhancers, and otherexpression control elements. Exemplary regulatory sequences aredescribed in Goeddel; Gene Expression Technology:

Methods in Enzymology, Academic Press, San Diego, Calif. (1990). Forinstance, any of a wide variety of expression control sequences thatcontrol the expression of a DNA sequence when operatively linked to itmay be used in these vectors to express DNA sequences encoding anActRIIB polypeptide. Such useful expression control sequences, include,for example, the early and late promoters of SV40, tet promoter,adenovirus or cytomegalovirus immediate early promoter, RSV promoters,the lac system, the trp system, the TAC or TRC system, T7 promoter whoseexpression is directed by T7 RNA polymerase, the major operator andpromoter regions of phage lambda, the control regions for fd coatprotein, the promoter for 3-phosphoglycerate kinase or other glycolyticenzymes, the promoters of acid phosphatase, e.g., Pho5, the promoters ofthe yeast α-mating factors, the polyhedron promoter of the baculovirussystem and other sequences known to control the expression of genes ofprokaryotic or eukaryotic cells or their viruses, and variouscombinations thereof. It should be understood that the design of theexpression vector may depend on such factors as the choice of the hostcell to be transformed and/or the type of protein desired to beexpressed. Moreover, the vector's copy number, the ability to controlthat copy number and the expression of any other protein encoded by thevector, such as antibiotic markers, should also be considered.

A recombinant nucleic acid of the invention can be produced by ligatingthe cloned gene, or a portion thereof, into a vector suitable forexpression in either prokaryotic cells, eukaryotic cells (yeast, avian,insect or mammalian), or both. Expression vehicles for production of arecombinant ActRIIB polypeptide include plasmids and other vectors. Forinstance, suitable vectors include plasmids of the types: pBR322-derivedplasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derivedplasmids and pUC-derived plasmids for expression in prokaryotic cells,such as E. coli.

Some mammalian expression vectors contain both prokaryotic sequences tofacilitate the propagation of the vector in bacteria, and one or moreeukaryotic transcription units that are expressed in eukaryotic cells.The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2,pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples ofmammalian expression vectors suitable for transfection of eukaryoticcells. Some of these vectors are modified with sequences from bacterialplasmids, such as pBR322, to facilitate replication and drug resistanceselection in both prokaryotic and eukaryotic cells. Alternatively,derivatives of viruses such as the bovine papilloma virus (BPV-1), orEpstein-Barr virus (pHEBo, pREP-derived and p205) can be used fortransient expression of proteins in eukaryotic cells. Examples of otherviral (including retroviral) expression systems can be found below inthe description of gene therapy delivery systems. The various methodsemployed in the preparation of the plasmids and in transformation ofhost organisms are well known in the art. For other suitable expressionsystems for both prokaryotic and eukaryotic cells, as well as generalrecombinant procedures, see Molecular Cloning A Laboratory Manual, 2ndEd., ed. by Sambrook, Fritsch and Maniatis (Cold Spring HarborLaboratory Press, 1989) Chapters 16 and 17. In some instances, it may bedesirable to express the recombinant polypeptides by the use of abaculovirus expression system. Examples of such baculovirus expressionsystems include pVL-derived vectors (such as pVL1392, pVL1393 andpVL941), pAcUW-derived vectors (such as pAcUWl), and pBlueBac-derivedvectors (such as the β-gal containing pBlueBac III).

In a preferred embodiment, a vector will be designed for production ofthe subject ActRIIB polypeptides in CHO cells, such as a Pcmv-Scriptvector (Stratagene, La Jolla, Calif), pcDNA4 vectors (Invitrogen,Carlsbad, Calif.) and pCI-neo vectors (Promega, Madison, Wis.). As willbe apparent, the subject gene constructs can be used to cause expressionof the subject ActRIIB polypeptides in cells propagated in culture,e.g., to produce proteins, including fusion proteins or variantproteins, for purification.

This invention also pertains to a host cell transfected with arecombinant gene including a coding sequence (e.g., SEQ ID NO: 4) forone or more of the subject ActRIIB polypeptide. The host cell may be anyprokaryotic or eukaryotic cell. For example, an ActRIIB polypeptide ofthe invention may be expressed in bacterial cells such as E. coli,insect cells (e.g., using a baculovirus expression system), yeast, ormammalian cells. Other suitable host cells are known to those skilled inthe art.

Accordingly, the present invention further pertains to methods ofproducing the subject ActRIIB polypeptides. For example, a host celltransfected with an expression vector encoding an ActRIIB polypeptidecan be cultured under appropriate conditions to allow expression of theActRIIB polypeptide to occur. The ActRIIB polypeptide may be secretedand isolated from a mixture of cells and medium containing the ActRIIBpolypeptide. Alternatively, the ActRIIB polypeptide may be retainedcytoplasmically or in a membrane fraction and the cells harvested, lysedand the protein isolated. A cell culture includes host cells, media andother byproducts. Suitable media for cell culture are well known in theart. The subject ActRIIB polypeptides can be isolated from cell culturemedium, host cells, or both, using techniques known in the art forpurifying proteins, including ion-exchange chromatography, gelfiltration chromatography, ultrafiltration, electrophoresis, andimmunoaffinity purification with antibodies specific for particularepitopes of the ActRIIB polypeptides. In a preferred embodiment, theActRIIB polypeptide is a fusion protein containing a domain whichfacilitates its purification.

In another embodiment, a fusion gene coding for a purification leadersequence, such as a poly-(His)/enterokinase cleavage site sequence atthe N-terminus of the desired portion of the recombinant ActRIIBpolypeptide, can allow purification of the expressed fusion protein byaffinity chromatography using a Ni²⁺ metal resin. The purificationleader sequence can then be subsequently removed by treatment withenterokinase to provide the purified ActRIIB polypeptide (e.g., seeHochuli et al., (1987) J. Chromatography 411:177; and Janknecht et al.,PNAS USA 88:8972).

Techniques for making fusion genes are well known. Essentially, thejoining of various DNA fragments coding for different polypeptidesequences is performed in accordance with conventional techniques,employing blunt-ended or stagger-ended termini for ligation, restrictionenzyme digestion to provide for appropriate termini, filling-in ofcohesive ends as appropriate, alkaline phosphatase treatment to avoidundesirable joining, and enzymatic ligation. In another embodiment, thefusion gene can be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which cansubsequently be annealed to generate a chimeric gene sequence (see, forexample, Current Protocols in Molecular Biology, eds. Ausubel et al.,John Wiley & Sons: 1992).

4. Antibodies

Another aspect of the invention pertains to antibodies. An antibody thatis specifically reactive with an ActRIIB polypeptide (e.g., a solubleActRIIB polypeptide) and which binds competitively with the ActRIIBpolypeptide may be used as an antagonist of ActRIIB polypeptideactivities. For example, by using immunogens derived from an ActRIIBpolypeptide, anti-protein/anti-peptide antisera or monoclonal antibodiescan be made by standard protocols (see, for example, Antibodies: ALaboratory Manual ed. by Harlow and Lane (Cold Spring Harbor Press:1988)). A mammal, such as a mouse, a hamster or rabbit can be immunizedwith an immunogenic form of the ActRIIB polypeptide, an antigenicfragment which is capable of eliciting an antibody response, or a fusionprotein. Techniques for conferring immunogenicity on a protein orpeptide include conjugation to carriers or other techniques well knownin the art. An immunogenic portion of an ActRIIB polypeptide can beadministered in the presence of adjuvant. The progress of immunizationcan be monitored by detection of antibody titers in plasma or serum.Standard ELISA or other immunoassays can be used with the immunogen asantigen to assess the levels of antibodies.

Following immunization of an animal with an antigenic preparation of anActRIIB polypeptide, antisera can be obtained and, if desired,polyclonal antibodies can be isolated from the serum. To producemonoclonal antibodies, antibody-producing cells (lymphocytes) can beharvested from an immunized animal and fused by standard somatic cellfusion procedures with immortalizing cells such as myeloma cells toyield hybridoma cells. Such techniques are well known in the art, andinclude, for example, the hybridoma technique (originally developed byKohler and Milstein, (1975) Nature, 256: 495-497), the human B cellhybridoma technique (Kozbar et al., (1983) Immunology Today, 4: 72), andthe EBV-hybridoma technique to produce human monoclonal antibodies (Coleet al., (1985) Monoclonal Antibodies and Cancer Therapy, Alan R. Liss,Inc. pp. 77-96). Hybridoma cells can be screened immunochemically forproduction of antibodies specifically reactive with an ActRIIBpolypeptide and monoclonal antibodies isolated from a culture comprisingsuch hybridoma cells.

The term “antibody” as used herein is intended to include fragmentsthereof which are also specifically reactive with a subject ActRIIBpolypeptide. Antibodies can be fragmented using conventional techniquesand the fragments screened for utility in the same manner as describedabove for whole antibodies. For example, F(ab)₂ fragments can begenerated by treating antibody with pepsin. The resulting F(ab)₂fragment can be treated to reduce disulfide bridges to produce Fabfragments. The antibody of the present invention is further intended toinclude bispecific, single-chain, and chimeric and humanized moleculeshaving affinity for an ActRIIB polypeptide conferred by at least one CDRregion of the antibody. In preferred embodiments, the antibody furthercomprises a label attached thereto and able to be detected (e.g., thelabel can be a radioisotope, fluorescent compound, enzyme or enzymeco-factor).

In certain preferred embodiments, an antibody of the invention is amonoclonal antibody, and in certain embodiments, the invention makesavailable methods for generating novel antibodies. For example, a methodfor generating a monoclonal antibody that binds specifically to anActRIIB polypeptide may comprise administering to a mouse an amount ofan immunogenic composition comprising the ActRIIB polypeptide effectiveto stimulate a detectable immune response, obtaining antibody-producingcells (e.g., cells from the spleen) from the mouse and fusing theantibody-producing cells with myeloma cells to obtain antibody-producinghybridomas, and testing the antibody-producing hybridomas to identify ahybridoma that produces a monoclonal antibody that binds specifically tothe ActRIIB polypeptide. Once obtained, a hybridoma can be propagated ina cell culture, optionally in culture conditions where thehybridoma-derived cells produce the monoclonal antibody that bindsspecifically to the ActRIIB polypeptide. The monoclonal antibody may bepurified from the cell culture.

The adjective “specifically reactive with” as used in reference to anantibody is intended to mean, as is generally understood in the art,that the antibody is sufficiently selective between the antigen ofinterest (e.g., an ActRIIB polypeptide) and other antigens that are notof interest that the antibody is useful for, at minimum, detecting thepresence of the antigen of interest in a particular type of biologicalsample. In certain methods employing the antibody, such as therapeuticapplications, a higher degree of specificity in binding may bedesirable. Monoclonal antibodies generally have a greater tendency (ascompared to polyclonal antibodies) to discriminate effectively betweenthe desired antigens and cross-reacting polypeptides. One characteristicthat influences the specificity of an antibody:antigen interaction isthe affinity of the antibody for the antigen. Although the desiredspecificity may be reached with a range of different affinities,generally preferred antibodies will have an affinity (a dissociationconstant) of about 10⁻⁶, 10⁻⁷, 10⁻⁸, 10⁻⁹ or less.

In addition, the techniques used to screen antibodies in order toidentify a desirable antibody may influence the properties of theantibody obtained. For example, if an antibody is to be used for bindingan antigen in solution, it may be desirable to test solution binding. Avariety of different techniques are available for testing interactionbetween antibodies and antigens to identify particularly desirableantibodies. Such techniques include ELISAs, surface plasmon resonancebinding assays (e.g., the Biacore binding assay, Bia-core AB, Uppsala,Sweden), sandwich assays (e.g., the paramagnetic bead system of IGENInternational, Inc., Gaithersburg, Md.), western blots,immunoprecipitation assays, and immunohistochemistry.

In certain aspects, the disclosure provides antibodies that bind to asoluble ActRIIB polypeptide. Such antibodies may be generated much asdescribed above, using a soluble ActRIIB polypeptide or fragment thereofas an antigen. Antibodies of this type can be used, e.g., to detectActRIIB polypeptides in biological samples and/or to monitor solubleActRIIB polypeptide levels in an individual. In certain cases, anantibody that specifically binds to a soluble ActRIIB polypeptide can beused to modulate activity of an ActRIIB polypeptide and/or an ActRIIBligand, thereby regulating (promoting or inhibiting) growth of tissues,such as bone, cartilage, muscle, fat, and neurons.

5. Screening Assays

In certain aspects, the present invention relates to the use of thesubject ActRIIB polypeptides (e.g., soluble ActRIIB polypeptides) toidentify compounds (agents) which are agonist or antagonists of theActRIIB polypeptides. Compounds identified through this screening can betested in tissues such as bone, cartilage, muscle, fat, and/or neurons,to assess their ability to modulate tissue growth in vitro. Optionally,these compounds can further be tested in animal models to assess theirability to modulate tissue growth in vivo.

There are numerous approaches to screening for therapeutic agents formodulating tissue growth by targeting the ActRIIB polypeptides. Incertain embodiments, high-throughput screening of compounds can becarried out to identify agents that perturb ActRIIB-mediated effects ongrowth of bone, cartilage, muscle, fat, and/or neurons. In certainembodiments, the assay is carried out to screen and identify compoundsthat specifically inhibit or reduce binding of an ActRIIB polypeptide toits binding partner, such as an ActRIIB ligand (e.g., activin, Nodal,GDF8, GDF11 or BMP7). Alternatively, the assay can be used to identifycompounds that enhance binding of an ActRIIB polypeptide to its bindingprotein such as an ActRIIB ligand. In a further embodiment, thecompounds can be identified by their ability to interact with an ActRIIBpolypeptide.

A variety of assay formats will suffice and, in light of the presentdisclosure, those not expressly described herein will nevertheless becomprehended by one of ordinary skill in the art. As described herein,the test compounds (agents) of the invention may be created by anycombinatorial chemical method. Alternatively, the subject compounds maybe naturally occurring biomolecules synthesized in vivo or in vitro.Compounds (agents) to be tested for their ability to act as modulatorsof tissue growth can be produced, for example, by bacteria, yeast,plants or other organisms (e.g., natural products), produced chemically(e.g., small molecules, including peptidomimetics), or producedrecombinantly. Test compounds contemplated by the present inventioninclude non-peptidyl organic molecules, peptides, polypeptides,peptidomimetics, sugars, hormones, and nucleic acid molecules. In aspecific embodiment, the test agent is a small organic molecule having amolecular weight of less than about 2,000 daltons.

The test compounds of the invention can be provided as single, discreteentities, or provided in libraries of greater complexity, such as madeby combinatorial chemistry. These libraries can comprise, for example,alcohols, alkyl halides, amines, amides, esters, aldehydes, ethers andother classes of organic compounds. Presentation of test compounds tothe test system can be in either an isolated form or as mixtures ofcompounds, especially in initial screening steps. Optionally, thecompounds may be optionally derivatized with other compounds and havederivatizing groups that facilitate isolation of the compounds.Non-limiting examples of derivatizing groups include biotin,fluorescein, digoxygenin, green fluorescent protein, isotopes,polyhistidine, magnetic beads, glutathione S transferase (GST),photoactivatable crosslinkers or any combinations thereof.

In many drug screening programs which test libraries of compounds andnatural extracts, high throughput assays are desirable in order tomaximize the number of compounds surveyed in a given period of time.Assays which are performed in cell-free systems, such as may be derivedwith purified or semi-purified proteins, are often preferred as“primary” screens in that they can be generated to permit rapiddevelopment and relatively easy detection of an alteration in amolecular target which is mediated by a test compound. Moreover, theeffects of cellular toxicity or bioavailability of the test compound canbe generally ignored in the in vitro system, the assay instead beingfocused primarily on the effect of the drug on the molecular target asmay be manifest in an alteration of binding affinity between an ActRIIBpolypeptide and its binding protein (e.g., an ActRIIB ligand).

Merely to illustrate, in an exemplary screening assay of the presentinvention, the compound of interest is contacted with an isolated andpurified ActRIIB polypeptide which is ordinarily capable of binding toan ActRIIB ligand, as appropriate for the intention of the assay. To themixture of the compound and ActRIIB polypeptide is then added acomposition containing an ActRIIB ligand. Detection and quantificationof ActRIIB/ActRIIB ligand complexes provides a means for determining thecompound's efficacy at inhibiting (or potentiating) complex formationbetween the ActRIIB polypeptide and its binding protein. The efficacy ofthe compound can be assessed by generating dose response curves fromdata obtained using various concentrations of the test compound.Moreover, a control assay can also be performed to provide a baselinefor comparison. For example, in a control assay, isolated and purifiedActRIIB ligand is added to a composition containing the ActRIIBpolypeptide, and the formation of ActRIIB/ActRIIB ligand complex isquantitated in the absence of the test compound. It will be understoodthat, in general, the order in which the reactants may be admixed can bevaried, and can be admixed simultaneously. Moreover, in place ofpurified proteins, cellular extracts and lysates may be used to render asuitable cell-free assay system.

Complex formation between the ActRIIB polypeptide and its bindingprotein may be detected by a variety of techniques. For instance,modulation of the formation of complexes can be quantitated using, forexample, detectably labeled proteins such as radiolabeled (e.g., ³²P,³⁵S, ¹⁴C or ³H), fluorescently labeled (e.g., FITC), or enzymaticallylabeled ActRIIB polypeptide or its binding protein, by immunoassay, orby chromatographic detection.

In certain embodiments, the present invention contemplates the use offluorescence polarization assays and fluorescence resonance energytransfer (FRET) assays in measuring, either directly or indirectly, thedegree of interaction between an ActRIIB polypeptide and its bindingprotein. Further, other modes of detection, such as those based onoptical waveguides (PCT Publication WO 96/26432 and U.S. Pat. No.5,677,196), surface plasmon resonance (SPR), surface charge sensors, andsurface force sensors, are compatible with many embodiments of theinvention.

Moreover, the present invention contemplates the use of an interactiontrap assay, also known as the “two hybrid assay,” for identifying agentsthat disrupt or potentiate interaction between an ActRIIB polypeptideand its binding protein. See for example, U.S. Pat. No. 5,283,317;Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J Biol Chem268:12046-12054; Bartel et al. (1993) Biotechniques 14:920-924; andIwabuchi et al. (1993) Oncogene 8:1693-1696). In a specific embodiment,the present invention contemplates the use of reverse two hybrid systemsto identify compounds (e.g., small molecules or peptides) thatdissociate interactions between an ActRIIB polypeptide and its bindingprotein. See for example, Vidal and Legrain, (1999) Nucleic Acids Res27:919-29; Vidal and Legrain, (1999) Trends Biotechnol 17:374-81; andU.S. Pat. Nos. 5,525,490; 5,955,280; and 5,965,368.

In certain embodiments, the subject compounds are identified by theirability to interact with an ActRIIB polypeptide of the invention. Theinteraction between the compound and the ActRIIB polypeptide may becovalent or non-covalent. For example, such interaction can beidentified at the protein level using in vitro biochemical methods,including photo-crosslinking, radiolabeled ligand binding, and affinitychromatography (Jakoby W B et al., 1974, Methods in Enzymology 46: 1).In certain cases, the compounds may be screened in a mechanism basedassay, such as an assay to detect compounds which bind to an ActRIIBpolypeptide. This may include a solid phase or fluid phase bindingevent. Alternatively, the gene encoding an ActRIIB polypeptide can betransfected with a reporter system (e.g., β-galactosidase, luciferase,or green fluorescent protein) into a cell and screened against thelibrary preferably by a high throughput screening or with individualmembers of the library. Other mechanism based binding assays may beused, for example, binding assays which detect changes in free energy.Binding assays can be performed with the target fixed to a well, bead orchip or captured by an immobilized antibody or resolved by capillaryelectrophoresis. The bound compounds may be detected usually usingcolorimetric or fluorescence or surface plasmon resonance.

In certain aspects, the present invention provides methods and agentsfor stimulating muscle growth and increasing muscle mass, for example,by antagonizing functions of an ActRIIB polypeptide and/or an ActRIIBligand. Therefore, any compound identified can be tested in whole cellsor tissues, in vitro or in vivo, to confirm their ability to modulatemuscle growth. Various methods known in the art can be utilized for thispurpose. For example, methods of the invention are performed such thatthe signal transduction through an ActRIIB protein activated by bindingto an ActRIIB ligand (e.g., GDF8) has been reduced or inhibited. It willbe recognized that the growth of muscle tissue in the organism wouldresult in an increased muscle mass in the organism as compared to themuscle mass of a corresponding organism (or population of organisms) inwhich the signal transduction through an ActRIIB protein had not been soeffected.

For example, the effect of the ActRIIB polypeptides or test compounds onmuscle cell growth/proliferation can be determined by measuring geneexpression of Pax-3 and Myf-5 which are associated with proliferation ofmyogenic cells, and gene expression of MyoD which is associated withmuscle differentiation (e.g., Amthor et al., Dev Biol. 2002,251:241-57). It is known that GDF8 down-regulates gene expression ofPax-3 and Myf-5, and prevents gene expression of MyoD. The ActRIIBpolypeptides or test compounds are expected to antagonize this activityof GDF8. Another example of cell-based assays includes measuring theproliferation of myoblasts such as C(2)C(12) myoblasts in the presenceof the ActRIIB polypeptides or test compounds (e.g., Thomas et al., JBiol Chem. 2000, 275:40235-43).

The present invention also contemplates in vivo assays to measure musclemass and strength. For example, Whittemore et al. (Biochem Biophys ResCommun. 2003, 300:965-71) discloses a method of measuring increasedskeletal muscle mass and increased grip strength in mice. Optionally,this method can be used to determine therapeutic effects of testcompounds (e.g., ActRIIB polypeptides) on muscle diseases or conditions,for example those diseases for which muscle mass is limiting.

In certain aspects, the present invention provides methods and agentsfor modulating (stimulating or inhibiting) bone formation and increasingbone mass. Therefore, any compound identified can be tested in wholecells or tissues, in vitro or in vivo, to confirm their ability tomodulate bone or cartilage growth. Various methods known in the art canbe utilized for this purpose.

For example, the effect of the ActRIIB polypeptides or test compounds onbone or cartilage growth can be determined by measuring induction ofMsx2 or differentiation of osteoprogenitor cells into osteoblasts incell based assays (see, e.g., Daluiski et al., Nat Genet. 2001,27(1):84-8; Hino et al., Front Biosci. 2004, 9:1520-9). Another exampleof cell-based assays includes analyzing the osteogenic activity of thesubject ActRIIB polypeptides and test compounds in mesenchymalprogenitor and osteoblastic cells. To illustrate, recombinantadenoviruses expressing an ActRIIB polypeptide were constructed toinfect pluripotent mesenchymal progenitor C3H10T1/2 cells,preosteoblastic C2C12 cells, and osteoblastic TE-85 cells. Osteogenicactivity is then determined by measuring the induction of alkalinephosphatase, osteocalcin, and matrix mineralization (see, e.g., Cheng etal., J bone Joint Surg Am. 2003, 85-A(8):1544-52).

The present invention also contemplates in vivo assays to measure boneor cartilage growth. For example, Namkung-Matthai et al., Bone, 28:80-86(2001) discloses a rat osteoporotic model in which bone repair duringthe early period after fracture is studied. Kubo et al., SteroidBiochemistry & Molecular Biology, 68:197-202 (1999) also discloses a ratosteoporotic model in which bone repair during the late period afterfracture is studied. These references are incorporated by referenceherein in their entirety for their disclosure of rat model for study onosteoporotic bone fracture. In certain aspects, the present inventionmakes use of fracture healing assays that are known in the art. Theseassays include fracture technique, histological analysis, andbiomechanical analysis, which are described in, for example, U.S. Pat.No. 6,521,750, which is incorporated by reference in its entirety forits disclosure of experimental protocols for causing as well asmeasuring the extent of fractures, and the repair process.

In certain aspects, the present invention provides methods and agentsfor controlling weight gain and obesity. At the cellular level,adipocyte proliferation and differentiation is critical in thedevelopment of obesity, which leads to the generation of additional fatcells (adipocytes). Therefore, any compound identified can be tested inwhole cells or tissues, in vitro or in vivo, to confirm their ability tomodulate adipogenesis by measuring adipocyte proliferation ordifferentiation. Various methods known in the art can be utilized forthis purpose. For example, the effect of an ActRIIB polypeptide (e.g., asoluble ActRIIB polypeptide) or test compounds on adipogenesis can bedetermined by measuring differentiation of 3T3-L1 preadipocytes tomature adipocytes in cell based assays, such as, by observing theaccumulation of triacylglycerol in Oil Red 0 staining vesicles and bythe appearance of certain adipocyte markers such as FABP (aP2/422) andPPARy2. See, for example, Reusch et al., 2000, Mol Cell Biol.20:1008-20; Deng et al., 2000, Endocrinology. 141:2370-6; Bell et al.,2000, Obes Res. 8:249-54. Another example of cell-based assays includesanalyzing the role of ActRIIB polypeptides and test compounds inproliferation of adipocytes or adipocyte precursor cells (e.g., 3T3-L1cells), such as, by monitoring bromodeoxyuridine (BrdU)-positive cells.See, for example, Pico et al., 1998, Mol Cell Biochem. 189:1-7; Masunoet al., 2003, Toxicol Sci. 75:314-20.

It is understood that the screening assays of the present inventionapply to not only the subject ActRIIB polypeptides and variants of theActRIIB polypeptides, but also any test compounds including agonists andantagonist of the ActRIIB polypeptides. Further, these screening assaysare useful for drug target verification and quality control purposes.

6. Exemplary Therapeutic Uses

In certain embodiments, compositions (e.g., ActRIIB polypeptides) of thepresent invention can be used for treating or preventing a disease orcondition that is associated with abnormal activity of an ActRIIBpolypeptide and/or an ActRIIB ligand (e.g., GDF8). These diseases,disorders or conditions are generally referred to herein as“ActRIIB-associated conditions.” In certain embodiments, the presentinvention provides methods of treating or preventing an individual inneed thereof through administering to the individual a therapeuticallyeffective amount of an ActRIIB polypeptide as described above. Thesemethods are particularly aimed at therapeutic and prophylactictreatments of animals, and more particularly, humans.

As used herein, a therapeutic that “prevents” a disorder or conditionrefers to a compound that, in a statistical sample, reduces theoccurrence of the disorder or condition in the treated sample relativeto an untreated control sample, or delays the onset or reduces theseverity of one or more symptoms of the disorder or condition relativeto the untreated control sample. The term “treating” as used hereinincludes prophylaxis of the named condition or amelioration orelimination of the condition once it has been established.

ActRIIB/ActRIIB ligand complexes play essential roles in tissue growthas well as early developmental processes such as the correct formationof various structures or in one or more post-developmental capacitiesincluding sexual development, pituitary hormone production, and creationof bone and cartilage. Thus, ActRIIB-associated conditions includeabnormal tissue growth and developmental defects. In addition,ActRIIB-associated conditions include, but are not limited to, disordersof cell growth and differentiation such as inflammation, allergy,autoimmune diseases, infectious diseases, and tumors.

Exemplary ActRIIB-associated conditions include neuromuscular disorders(e.g., muscular dystrophy and muscle atrophy), congestive obstructivepulmonary disease (and muscle wasting associated with COPD), musclewasting syndrome, sarcopenia, cachexia, adipose tissue disorders (e.g.,obesity), type 2 diabetes, and bone degenerative disease (e.g.,osteoporosis). Other exemplary ActRIIB-associated conditions includemusculodegenerative and neuromuscular disorders, tissue repair (e.g.,wound healing), neurodegenerative diseases (e.g., amyotrophic lateralsclerosis), immunologic disorders (e.g., disorders related to abnormalproliferation or function of lymphocytes), and obesity or disordersrelated to abnormal proliferation of adipocytes.

In certain embodiments, compositions (e.g., soluble ActRIIBpolypeptides) of the invention are used as part of a treatment for amuscular dystrophy. The term “muscular dystrophy” refers to a group ofdegenerative muscle diseases characterized by gradual weakening anddeterioration of skeletal muscles and sometimes the heart andrespiratory muscles. Muscular dystrophies are genetic disorderscharacterized by progressive muscle wasting and weakness that begin withmicroscopic changes in the muscle. As muscles degenerate over time, theperson's muscle strength declines. Exemplary muscular dystrophies thatcan be treated with a regimen including the subject ActRIIB polypeptidesinclude: Duchenne Muscular Dystrophy (DMD), Becker Muscular Dystrophy(BMD), Emery-Dreifuss Muscular Dystrophy (EDMD), Limb-Girdle MuscularDystrophy (LGMD), Facioscapulohumeral Muscular Dystrophy (FSH or FSHD)(also known as Landouzy-Dejerine), Myotonic Dystrophy (MMD) (also knownas Steinert's Disease), Oculopharyngeal Muscular Dystrophy (OPMD),Distal Muscular Dystrophy (DD), Congenital Muscular Dystrophy (CMD).

Duchenne Muscular Dystrophy (DMD) was first described by the Frenchneurologist Guillaume Benjamin Amand Duchenne in the 1860s. BeckerMuscular Dystrophy (BMD) is named after the German doctor Peter EmilBecker, who first described this variant of DMD in the 1950s. DMD is oneof the most frequent inherited diseases in males, affecting one in 3,500boys. DMD occurs when the dystrophin gene, located on the short arm ofthe X chromosome, is broken. Since males only carry one copy of the Xchromosome, they only have one copy of the dystrophin gene. Without thedystrophin protein, muscle is easily damaged during cycles ofcontraction and relaxation. While early in the disease musclecompensates by regeneration, later on muscle progenitor cells cannotkeep up with the ongoing damage and healthy muscle is replaced bynon-functional fibro-fatty tissue.

BMD results from different mutations in the dystrophin gene. BMDpatients have some dystrophin, but it is either insufficient in quantityor poor in quality. Having some dystrophin protects the muscles of thosewith BMD from degenerating as badly or as quickly as those of peoplewith DMD.

For example, recent researches demonstrate that blocking or eliminatingfunction of GDF8 (an ActRIIB ligand) in vivo can effectively treat atleast certain symptoms in DMD and BMD patients. Thus, the subjectActRIIB polypeptides may act as GDF8 inhibitors (antagonists), andconstitute an alternative means of blocking the functions of GDF8 and/orActRIIB in vivo in DMD and BMD patients. This approach is confirmed andsupported by the data shown herein, whereby an ActRIIB-Fc protein wasshown to increase muscle mass in a mouse model of muscular dystrophy.

Similarly, the subject ActRIIB polypeptides provide an effective meansto increase muscle mass in other disease conditions that are in need ofmuscle growth. For example, ALS, also called Lou Gehrig's disease (motorneuron disease) is a chronic, incurable, and unstoppable CNS disorderthat attacks the motor neurons, components of the CNS that connect thebrain to the skeletal muscles. In ALS, the motor neurons deteriorate andeventually die, and though a person's brain normally remains fullyfunctioning and alert, the command to move never reaches the muscles.Most people who get ALS are between 40 and 70 years old. The first motorneurons that weaken are those leading to the arms or legs. Those withALS may have trouble walking, they may drop things, fall, slur theirspeech, and laugh or cry uncontrollably. Eventually the muscles in thelimbs begin to atrophy from disuse. This muscle weakness will becomedebilitating and a person will need a wheel chair or become unable tofunction out of bed. Most ALS patients die from respiratory failure orfrom complications of ventilator assistance like pneumonia, 3-5 yearsfrom disease onset. This approach is confirmed and supported by the datashown herein, whereby an ActRIIB-Fc protein was shown to improve theappearance, muscle mass and lifespan of a mouse model of ALS.

ActRIIB polypeptide-induced increased muscle mass might also benefitthose suffering from muscle wasting diseases. Gonzalez-Cadavid et al.(supra) reported that that GDF8 expression correlates inversely withfat-free mass in humans and that increased expression of the GDF8 geneis associated with weight loss in men with AIDS wasting syndrome. Byinhibiting the function of GDF8 in AIDS patients, at least certainsymptoms of AIDS may be alleviated, if not completely eliminated, thussignificantly improving quality of life in AIDS patients.

Since loss of GDF8 (an ActRIIB ligand) function is also associated withfat loss without diminution of nutrient intake (Zimmers et al., supra;McPherron and Lee, supra), the subject ActRIIB polypeptides may furtherbe used as a therapeutic agent for slowing or preventing the developmentof obesity and type II diabetes. This approach is confirmed andsupported by the data shown herein, whereby an ActRIIB-Fc protein wasshown to improve metabolic status in obese mice.

The cancer anorexia-cachexia syndrome is among the most debilitating andlife-threatening aspects of cancer. Progressive weight loss in canceranorexia-cachexia syndrome is a common feature of many types of cancerand is responsible not only for a poor quality of life and poor responseto chemotherapy, but also a shorter survival time than is found inpatients with comparable tumors without weight loss. Associated withanorexia, fat and muscle tissue wasting, psychological distress, and alower quality of life, cachexia arises from a complex interactionbetween the cancer and the host. It is one of the most common causes ofdeath among cancer patients and is present in 80% at death. It is acomplex example of metabolic chaos effecting protein, carbohydrate, andfat metabolism. Tumors produce both direct and indirect abnormalities,resulting in anorexia and weight loss. Currently, there is no treatmentto control or reverse the process. Cancer anorexia-cachexia syndromeaffects cytokine production, release of lipid-mobilizing andproteolysis-inducing factors, and alterations in intermediarymetabolism. Although anorexia is common, a decreased food intake aloneis unable to account for the changes in body composition seen in cancerpatients, and increasing nutrient intake is unable to reverse thewasting syndrome. Cachexia should be suspected in patients with cancerif an involuntary weight loss of greater than five percent of premorbidweight occurs within a six-month period.

Since systemic overexpression of GDF8 in adult mice was found to induceprofound muscle and fat loss analogous to that seen in human cachexiasyndromes (Zimmers et al., supra), the subject ActRIIB polypeptides aspharmaceutical compositions can be beneficially used to prevent, treat,or alleviate the symptoms of the cachexia syndrome, where muscle growthis desired.

In other embodiments, the present invention provides methods of inducingbone and/or cartilage formation, preventing bone loss, increasing bonemineralization or preventing the demineralization of bone. For example,the subject ActRIIB polypeptides and compounds identified in the presentinvention have application in treating osteoporosis and the healing ofbone fractures and cartilage defects in humans and other animals.ActRIIB polypeptides may be useful in patients that are diagnosed withsubclinical low bone density, as a protective measure against thedevelopment of osteoporosis.

In one specific embodiment, methods and compositions of the presentinvention may find medical utility in the healing of bone fractures andcartilage defects in humans and other animals. The subject methods andcompositions may also have prophylactic use in closed as well as openfracture reduction and also in the improved fixation of artificialjoints. De novo bone formation induced by an osteogenic agentcontributes to the repair of congenital, trauma-induced, or oncologicresection induced craniofacial defects, and also is useful in cosmeticplastic surgery. Further, methods and compositions of the invention maybe used in the treatment of periodontal disease, and in other toothrepair processes. In certain cases, the subject ActRIIB polypeptides mayprovide an environment to attract bone-forming cells, stimulate growthof bone-forming cells or induce differentiation of progenitors ofbone-forming cells. ActRIIB polypeptides of the invention may also beuseful in the treatment of osteoporosis. Further, ActRIIB polypeptidesmay be used in cartilage defect repair and prevention/reversal ofosteoarthritis.

In another specific embodiment, the invention provides a therapeuticmethod and composition for repairing fractures and other conditionsrelated to cartilage and/or bone defects or periodontal diseases. Theinvention further provides therapeutic methods and compositions forwound healing and tissue repair. The types of wounds include, but arenot limited to, burns, incisions and ulcers. See e.g., PCT PublicationNo. WO84/01106. Such compositions comprise a therapeutically effectiveamount of at least one of the ActRIIB polypeptides of the invention inadmixture with a pharmaceutically acceptable vehicle, carrier or matrix.

In another specific embodiment, methods and compositions of theinvention can be applied to conditions causing bone loss such asosteoporosis, hyperparathyroidism, Cushing's disease, thyrotoxicosis,chronic diarrheal state or malabsorption, renal tubular acidosis, oranorexia nervosa. Many people know that being female, having a low bodyweight, and leading a sedentary lifestyle are risk factors forosteoporosis (loss of bone mineral density, leading to fracture risk).However, osteoporosis can also result from the long-term use of certainmedications. Osteoporosis resulting from drugs or another medicalcondition is known as secondary osteoporosis. In a condition known asCushing's disease, the excess amount of cortisol produced by the bodyresults in osteoporosis and fractures. The most common medicationsassociated with secondary osteoporosis are the corticosteroids, a classof drugs that act like cortisol, a hormone produced naturally by theadrenal glands. Although adequate levels of thyroid hormones (which areproduced by the thyroid gland) are needed for the development of theskeleton, excess thyroid hormone can decrease bone mass over time.Antacids that contain aluminum can lead to bone loss when taken in highdoses by people with kidney problems, particularly those undergoingdialysis. Other medications that can cause secondary osteoporosisinclude phenytoin (Dilantin) and barbiturates that are used to preventseizures; methotrexate (Rheumatrex, Immunex, Folex PFS), a drug for someforms of arthritis, cancer, and immune disorders; cyclosporine(Sandimmune, Neoral), a drug used to treat some autoimmune diseases andto suppress the immune system in organ transplant patients; luteinizinghormone-releasing hormone agonists (Lupron, Zoladex), used to treatprostate cancer and endometriosis; heparin (Calciparine, Liquaemin), ananticlotting medication; and cholestyramine (Questran) and colestipol(Colestid), used to treat high cholesterol. Gum disease causes bone lossbecause these harmful bacteria in our mouths force our bodies to defendagainst them. The bacteria produce toxins and enzymes under thegum-line, causing a chronic infection.

In a further embodiment, the present invention provides methods andtherapeutic agents for treating diseases or disorders associated withabnormal or unwanted bone growth. For example, patients having thedisease known as Fibrodysplasia Ossificans Progressiva (FOP) grow anabnormal “second skeleton” that prevents any movement. Additionally,abnormal bone growth can occur after hip replacement surgery and thusruin the surgical outcome. This is a more common example of pathologicalbone growth and a situation in which the subject methods andcompositions may be therapeutically useful. The same methods andcompositions may also be useful for treating other forms of abnormalbone growth (e.g., pathological growth of bone following trauma, burnsor spinal cord injury), and for treating or preventing the undesirableconditions associated with the abnormal bone growth seen in connectionwith metastatic prostate cancer or osteosarcoma. Examples of thesetherapeutic agents include, but are not limited to, ActRIIB polypeptidesthat antagonize function of an ActRIIB ligand (e.g., BMP7), compoundsthat disrupt interaction between an ActRIIB and its ligand (e.g., BMP7),and antibodies that specifically bind to an ActRIIB receptor such thatan ActRIIB ligand (e.g., BMP7) cannot bind to the ActRIIB receptor.

In other embodiments, the present invention provides compositions andmethods for regulating body fat content in an animal and for treating orpreventing conditions related thereto, and particularly,health-compromising conditions related thereto. According to the presentinvention, to regulate (control) body weight can refer to reducing orincreasing body weight, reducing or increasing the rate of weight gain,or increasing or reducing the rate of weight loss, and also includesactively maintaining, or not significantly changing body weight (e.g.,against external or internal influences which may otherwise increase ordecrease body weight). One embodiment of the present invention relatesto regulating body weight by administering to an animal (e.g., a human)in need thereof an ActRIIB polypeptide.

In one specific embodiment, the present invention relates to methods andcompounds for reducing body weight and/or reducing weight gain in ananimal, and more particularly, for treating or ameliorating obesity inpatients at risk for or suffering from obesity. In another specificembodiment, the present invention is directed to methods and compoundsfor treating an animal that is unable to gain or retain weight (e.g., ananimal with a wasting syndrome). Such methods are effective to increasebody weight and/or mass, or to reduce weight and/or mass loss, or toimprove conditions associated with or caused by undesirably low (e.g.,unhealthy) body weight and/or mass.

Other disorders, including high cholesterol, that may be treated withActRIIB proteins are described in the Examples.

7. Pharmaceutical Compositions

In certain embodiments, compounds (e.g., ActRIIB polypeptides) of thepresent invention are formulated with a pharmaceutically acceptablecarrier. For example, an ActRIIB polypeptide can be administered aloneor as a component of a pharmaceutical formulation (therapeuticcomposition). The subject compounds may be formulated for administrationin any convenient way for use in human or veterinary medicine.

In certain embodiments, the therapeutic method of the invention includesadministering the composition topically, systemically, or locally as animplant or device. When administered, the therapeutic composition foruse in this invention is, of course, in a pyrogen-free, physiologicallyacceptable form. Further, the composition may desirably be encapsulatedor injected in a viscous form for delivery to a target tissue site(e.g., bone, cartilage, muscle, fat or neurons), for example, a sitehaving a tissue damage. Topical administration may be suitable for woundhealing and tissue repair. Therapeutically useful agents other than theActRIIB polypeptides which may also optionally be included in thecomposition as described above, may alternatively or additionally, beadministered simultaneously or sequentially with the subject compounds(e.g., ActRIIB polypeptides) in the methods of the invention.

In certain embodiments, compositions of the present invention mayinclude a matrix capable of delivering one or more therapeutic compounds(e.g., ActRIIB polypeptides) to a target tissue site, providing astructure for the developing tissue and optimally capable of beingresorbed into the body. For example, the matrix may provide slow releaseof the ActRIIB polypeptides. Such matrices may be formed of materialspresently in use for other implanted medical applications.

The choice of matrix material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. The particular application of the subjectcompositions will define the appropriate formulation. Potential matricesfor the compositions may be biodegradable and chemically defined calciumsulfate, tricalciumphosphate, hydroxyapatite, polylactic acid andpolyanhydrides. Other potential materials are biodegradable andbiologically well defined, such as bone or dermal collagen. Furthermatrices are comprised of pure proteins or extracellular matrixcomponents. Other potential matrices are non-biodegradable andchemically defined, such as sintered hydroxyapatite, bioglass,aluminates, or other ceramics. Matrices may be comprised of combinationsof any of the above mentioned types of material, such as polylactic acidand hydroxyapatite or collagen and tricalciumphosphate. The bioceramicsmay be altered in composition, such as in calcium-aluminate-phosphateand processing to alter pore size, particle size, particle shape, andbiodegradability.

In certain embodiments, methods of the invention can be administered fororally, e.g., in the form of capsules, cachets, pills, tablets, lozenges(using a flavored basis, usually sucrose and acacia or tragacanth),powders, granules, or as a solution or a suspension in an aqueous ornon-aqueous liquid, or as an oil-in-water or water-in-oil liquidemulsion, or as an elixir or syrup, or as pastilles (using an inertbase, such as gelatin and glycerin, or sucrose and acacia) and/or asmouth washes and the like, each containing a predetermined amount of anagent as an active ingredient. An agent may also be administered as abolus, electuary or paste.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules, and the like), one or more therapeuticcompounds of the present invention may be mixed with one or morepharmaceutically acceptable carriers, such as sodium citrate ordicalcium phosphate, and/or any of the following: (1) fillers orextenders, such as starches, lactose, sucrose, glucose, mannitol, and/orsilicic acid; (2) binders, such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia; (3)humectants, such as glycerol; (4) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, and sodium carbonate; (5) solution retarding agents,such as paraffin; (6) absorption accelerators, such as quaternaryammonium compounds; (7) wetting agents, such as, for example, cetylalcohol and glycerol monostearate; (8) absorbents, such as kaolin andbentonite clay; (9) lubricants, such a talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, andmixtures thereof; and (10) coloring agents. In the case of capsules,tablets and pills, the pharmaceutical compositions may also comprisebuffering agents. Solid compositions of a similar type may also beemployed as fillers in soft and hard-filled gelatin capsules using suchexcipients as lactose or milk sugars, as well as high molecular weightpolyethylene glycols and the like.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups,and elixirs. In addition to the active ingredient, the liquid dosageforms may contain inert diluents commonly used in the art, such as wateror other solvents, solubilizing agents and emulsifiers, such as ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor, andsesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. Besides inertdiluents, the oral compositions can also include adjuvants such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming, and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents such as ethoxylated isostearyl alcohols, polyoxyethylenesorbitol, and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Certain compositions disclosed herein may be administered topically,either to skin or to mucosal membranes. The topical formulations mayfurther include one or more of the wide variety of agents known to beeffective as skin or stratum corneum penetration enhancers. Examples ofthese are 2-pyrrolidone, N-methyl-2-pyrrolidone, dimethylacetamide,dimethylformamide, propylene glycol, methyl or isopropyl alcohol,dimethyl sulfoxide, and azone. Additional agents may further be includedto make the formulation cosmetically acceptable. Examples of these arefats, waxes, oils, dyes, fragrances, preservatives, stabilizers, andsurface active agents. Keratolytic agents such as those known in the artmay also be included. Examples are salicylic acid and sulfur.

Dosage forms for the topical or transdermal administration includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches, and inhalants. The active compound may be mixed under sterileconditions with a pharmaceutically acceptable carrier, and with anypreservatives, buffers, or propellants which may be required. Theointments, pastes, creams and gels may contain, in addition to a subjectcompound of the invention (e.g., an ActRIIB polypeptide), excipients,such as animal and vegetable fats, oils, waxes, paraffins, starch,tragacanth, cellulose derivatives, polyethylene glycols, silicones,bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a subject compound,excipients such as lactose, talc, silicic acid, aluminum hydroxide,calcium silicates, and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

In certain embodiments, pharmaceutical compositions suitable forparenteral administration may comprise one or more ActRIIB polypeptidesin combination with one or more pharmaceutically acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents. Examples of suitable aqueous andnonaqueous carriers which may be employed in the pharmaceuticalcompositions of the invention include water, ethanol, polyols (such asglycerol, propylene glycol, polyethylene glycol, and the like), andsuitable mixtures thereof, vegetable oils, such as olive oil, andinjectable organic esters, such as ethyl oleate. Proper fluidity can bemaintained, for example, by the use of coating materials, such aslecithin, by the maintenance of the required particle size in the caseof dispersions, and by the use of surfactants.

The compositions of the invention may also contain adjuvants, such aspreservatives, wetting agents, emulsifying agents and dispersing agents.Prevention of the action of microorganisms may be ensured by theinclusion of various antibacterial and antifungal agents, for example,paraben, chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption, such as aluminum monostearate andgelatin.

It is understood that the dosage regimen will be determined by theattending physician considering various factors which modify the actionof the subject compounds of the invention (e.g., ActRIIB polypeptides).The various factors will depend upon the disease to be treated. In thecase of muscle disorders, factors may include, but are not limited to,amount of muscle mass desired to be formed, the muscles most affected bydisease, the condition of the deteriorated muscle, the patient's age,sex, and diet, time of administration, and other clinical factors. Theaddition of other known growth factors to the final composition, mayalso affect the dosage. Progress can be monitored by periodic assessmentof muscle growth and/or repair, for example, by strength testing, MRIassessment of muscle size and analysis of muscle biopsies.

In certain embodiments of the invention, one or more ActRIIBpolypeptides can be administered, together (simultaneously) or atdifferent times (sequentially or overlapping). In addition, ActRIIBpolypeptides can be administered with another type of therapeuticagents, for example, a cartilage-inducing agent, a bone-inducing agent,a muscle-inducing agent, a fat-reducing, or a neuron-inducing agent. Thetwo types of compounds may be administered simultaneously or atdifferent times. It is expected that the ActRIIB polypeptides of theinvention may act in concert with or perhaps synergistically withanother therapeutic agent.

In a specific example, a variety of osteogenic, cartilage-inducing andbone-inducing factors have been described, particularly bisphosphonates.See e.g., European Patent Application Nos. 148,155 and 169,016. Forexample, other factors that can be combined with the subject ActRIIBpolypeptides include various growth factors such as epidermal growthfactor (EGF), platelet derived growth factor (PDGF), transforming growthfactors (TGF-α and TGF-β), and insulin-like growth factor (IGF).

In certain embodiments, the present invention also provides gene therapyfor the in vivo production of ActRIIB polypeptides. Such therapy wouldachieve its therapeutic effect by introduction of the ActRIIBpolynucleotide sequences into cells or tissues having the disorders aslisted above. Delivery of ActRIIB polynucleotide sequences can beachieved using a recombinant expression vector such as a chimeric virusor a colloidal dispersion system. Preferred for therapeutic delivery ofActRIIB polynucleotide sequences is the use of targeted liposomes.

Various viral vectors which can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia, or, preferably, anRNA virus such as a retrovirus. Preferably, the retroviral vector is aderivative of a murine or avian retrovirus. Examples of retroviralvectors in which a single foreign gene can be inserted include, but arenot limited to: Moloney murine leukemia virus (MoMuLV), Harvey murinesarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and RousSarcoma Virus (RSV). A number of additional retroviral vectors canincorporate multiple genes. All of these vectors can transfer orincorporate a gene for a selectable marker so that transduced cells canbe identified and generated. Retroviral vectors can be madetarget-specific by attaching, for example, a sugar, a glycolipid, or aprotein. Preferred targeting is accomplished by using an antibody. Thoseof skill in the art will recognize that specific polynucleotidesequences can be inserted into the retroviral genome or attached to aviral envelope to allow target specific delivery of the retroviralvector containing the ActRIIB polynucleotide. In one preferredembodiment, the vector is targeted to bone, cartilage, muscle or neuroncells/tissues.

Alternatively, tissue culture cells can be directly transfected withplasmids encoding the retroviral structural genes gag, pol and env, byconventional calcium phosphate transfection. These cells are thentransfected with the vector plasmid containing the genes of interest.The resulting cells release the retroviral vector into the culturemedium.

Another targeted delivery system for ActRIIB polynucleotides is acolloidal dispersion system. Colloidal dispersion systems includemacromolecule complexes, nanocapsules, microspheres, beads, andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes. The preferred colloidal system of thisinvention is a liposome. Liposomes are artificial membrane vesicleswhich are useful as delivery vehicles in vitro and in vivo. RNA, DNA andintact virions can be encapsulated within the aqueous interior and bedelivered to cells in a biologically active form (see e.g., Fraley, etal., Trends Biochem. Sci., 6:77, 1981). Methods for efficient genetransfer using a liposome vehicle, are known in the art, see e.g.,Mannino, et al., Biotechniques, 6:682, 1988. The composition of theliposome is usually a combination of phospholipids, usually incombination with steroids, especially cholesterol. Other phospholipidsor other lipids may also be used. The physical characteristics ofliposomes depend on pH, ionic strength, and the presence of divalentcations.

Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides. Illustrative phospholipids include eggphosphatidylcholine, dipalmitoylphosphatidylcholine, anddistearoylphosphatidylcholine. The targeting of liposomes is alsopossible based on, for example, organ-specificity, cell-specificity, andorganelle-specificity and is known in the art.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain embodiments andembodiments of the present invention, and are not intended to limit theinvention.

Example 1. Generation of an ActRIIb-Fc Fusion Protein

Applicants constructed a soluble ActRIIb fusion protein that has theextracellular domain of human ActRIIb fused to a human or mouse Fcdomain with a minimal linker (three glycine amino acids) in between. Theconstructs are referred to as ActRIIb-hFc and ActRIIb-mFc, respectively.

ActRIIb-hFc is shown below as purified from CHO cell lines (SEQ ID NO:5)

GRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The ActRIIb-hFc and ActRIIb-mFc proteins were expressed in CHO celllines. Three different leader sequences were considered:

(i) Honey bee mellitin (HBML): (SEQ ID NO: 7) MKFLVNVALVFMVVYISYIYA(ii) Tissue Plasminogen Activator (TPA): (SEQ ID NO: 8)MDAMKRGLCCVLLLCGAVFVSP (iii) Native: (SEQ ID NO: 9)MGAAAKLAFAVFLISCSSGA.

The selected form employs the TPA leader and has the followingunprocessed amino acid sequence:

MDAMKRGLCCVLLLCGAVFVSPGASGRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

This polypeptide is encoded by the following nucleic acid sequence (SEQID NO:10):

A TGGATGCAAT GAAGAGAGGG CTCTGCTGTG TGCTGCTGCT GTGTGGAGCA GTCTTCGTTTCGCCCGGCGC CTCTGGGCGT GGGGAGGCTG AGACACGGGA GTGCATCTAC TACAACGCCAACTGGGAGCT GGAGCGCACC AACCAGAGCG GCCTGGAGCG CTGCGAAGGC GAGCAGGACAAGCGGCTGCA CTGCTACGCC TCCTGGCGCA ACAGCTCTGG CACCATCGAG CTCGTGAAGAAGGGCTGCTG GCTAGATGAC TTCAACTGCT ACGATAGGCA GGAGTGTGTG GCCACTGAGGAGAACCCCCA GGTGTACTTC TGCTGCTGTG AAGGCAACTT CTGCAACGAG CGCTTCACTCATTTGCCAGA GGCTGGGGGC CCGGAAGTCA CGTACGAGCC ACCCCCGACA GCCCCCACCGGTGGTGGAAC TCACACATGC CCACCGTGCC CAGCACCTGA ACTCCTGGGG GGACCGTCAGTCTTCCTCTT CCCCCCAAAA CCCAAGGACA CCCTCATGAT CTCCCGGACC CCTGAGGTCACATGCGTGGT GGTGGACGTG AGCCACGAAG ACCCTGAGGT CAAGTTCAAC TGGTACGTGGACGGCGTGGA GGTGCATAAT GCCAAGACAA AGCCGCGGGA GGAGCAGTAC AACAGCACGTACCGTGTGGT CAGCGTCCTC ACCGTCCTGC ACCAGGACTG GCTGAATGGC AAGGAGTACAAGTGCAAGGT CTCCAACAAA GCCCTCCCAG TCCCCATCGA GAAAACCATC TCCAAAGCCAAAGGGCAGCC CCGAGAACCA CAGGTGTACA CCCTGCCCCC ATCCCGGGAG GAGATGACCAAGAACCAGGT CAGCCTGACC TGCCTGGTCA AAGGCTTCTA TCCCAGCGAC ATCGCCGTGGAGTGGGAGAG CAATGGGCAG CCGGAGAACA ACTACAAGAC CACGCCTCCC GTGCTGGACTCCGACGGCTC CTTCTTCCTC TATAGCAAGC TCACCGTGGA CAAGAGCAGG TGGCAGCAGGGGAACGTCTT CTCATGCTCC GTGATGCATG AGGCTCTGCA CAACCACTAC ACGCAGAAGAGCCTCTCCCT GTCTCCGGGT AAATGA

N-terminal sequencing of the CHO-cell produced material revealed a majorsequence of -GRGEAE (SEQ ID NO: 11). Notably, other constructs reportedin the literature begin with an —SGR . . . sequence.

Purification could be achieved by a series of column chromatographysteps, including, for example, three or more of the following, in anyorder: protein A chromatography, Q sepharose chromatography,phenylsepharose chromatography, size exclusion chromatography, andcation exchange chromatography. The purification could be completed withviral filtration and buffer exchange.

ActRIIb-Fc fusion proteins were also expressed in HEK293 cells and COScells. Although material from all cell lines and reasonable cultureconditions provided protein with muscle-building activity in vivo,variability in potency was observed perhaps relating to cell lineselection and/or culture conditions.

Example 2: Generation of ActRIIb-Fc Mutants

Applicants generated a series of mutations in the extracellular domainof ActRIIB and produced these mutant proteins as soluble fusion proteinsbetween extracellular ActRIIB and an Fc domain. The backgroundActRIIB-Fc fusion has the sequence (Fc portion underlined)(SEQ IDNO:12):

SGRGEAETRECIYYNANWELERTNQSGLERCEGEQDKRLHCYASWRNSSGTIELVKKGCWLDDFNCYDRQECVATEENPQVYFCCCEGNFCNERFTHLPEAGGPEVTYEPPPTAPTGGGTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPVPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG K

Various mutations, including N- and C-terminal truncations, wereintroduced into the background ActRIIB-Fc protein. Based on the datapresented in Example 1, it is expected that these constructs, ifexpressed with a TPA leader, will lack the N-terminal serine. Mutationswere generated in ActRIIB extracellular domain by PCR mutagenesis. AfterPCR, fragments were purified through a Qiagen column, digested with SfoIand AgeI and gel purified. These fragments were ligated into expressionvector pAID4 (see WO2006/012627) such that upon ligation it createdfusion chimera with human IgG1. Upon transformation into E. coli DH5alpha, colonies were picked and DNAs were isolated. For murineconstructs (mFc), a murine IgG2a was substituted for the human IgG1. Allmutants were sequence verified.

All of the mutants were produced in HEK293T cells by transienttransfection. In summary, in a 500 ml spinner, HEK293T cells were set upat 6×10⁵ cells/ml in Freestyle (Invitrogen) media in 250 ml volume andgrown overnight. Next day, these cells were treated with DNA:PEI (1:1)complex at 0.5 ug/ml final DNA concentration. After 4 hrs, 250 ml mediawas added and cells were grown for 7 days. Conditioned media washarvested by spinning down the cells and concentrated.

Mutants were purified using a variety of techniques, including, forexample, protein A column and eluted with low pH (3.0) glycine buffer.After neutralization, these were dialyzed against PBS.

Mutants were also produced in CHO cells by similar methodology.

Mutants were tested in binding assays and/or bioassays described below.In some instances, assays were performed with conditioned medium ratherthan purified proteins.

Example 2. Bioassay for GDF-11 and Activin-Mediated Signaling

An A-204 Reporter Gene Assay was used to evaluate the effects ofActRIIB-Fc proteins on signaling by GDF-11 and Activin A. Cell line:Human Rhabdomyosarcoma (derived from muscle). Reporter vector:pGL3(CAGA)12 (Described in Dennler et al, 1998, EMBO 17: 3091-3100.) SeeFIG. 5. The CAGA12 motif is present in TGF-Beta responsive genes (PAI-1gene), so this vector is of general use for factors signaling throughSmad2 and 3.

Day 1: Split A-204 cells into 48-well plate.

Day 2: A-204 cells transfected with 10 ug pGL3(CAGA)12 or pGL3(CAGA)12(10 ug)+pRLCMV (1 ug) and Fugene.

Day 3: Add factors (diluted into medium+0.1% BSA). Inhibitors need to bepreincubated with Factors for 1 hr before adding to cells. 6 hrs later,cells rinsed with PBS, and lyse cells.

This is followed by a Luciferase assay. In the absence of anyinhibitors, Activin A showed 10 fold stimulation of reporter geneexpression and an ED50-2 ng/ml. GDF-11: 16 fold stimulation, ED50: ˜1.5ng/ml.

ActRIIB (R64, 20-134) is a potent inhibitor of activin, GDF-8 and GDF-11activity in this assay. Variants were tested in this assay as well.

Example 3. GDF-11 Inhibition of N-Terminal and C-Terminal Truncations

Truncations at the N-terminus and C-terminus of the ActRIIB portionActRIIB-Fc (R64, 20-134) were generated and tested for activity asinhibitors of GDF-11 and activin. The activities are shown below (asmeasured in conditioned media):

C-Terminal ActRIIb-hFc Truncations:

IC50 (ng/mL) GDF-11 Activin ActRIIb-hFc (R64, 20-134) 45 22 ActRIIb-hFc(R64, 20-132) 87 32 ActRIIb-hFc (R64, 20-131) 120 44 ActRIIb-hFc (R64,20-128) 130 158

As can be seen, truncations of three (ending with . . . PPT), six(ending with . . . YEP) or more amino acids at the C-terminus causes athreefold or greater decrease in the activity of the molecule. Thetruncation of the final 15 amino acids of the ActRIIB portion causes agreater loss of activity (see WO2006/012627).

Amino terminal truncations were made in the background of an ActRIIb-hFc(R64 20-131) protein. The activities are shown below (as measured inconditioned media):

N-Terminal ActRIIb-hFc Truncations:

IC50 (ng/mL) GDF-11 Activin ActRIIb-hFc (R64, 20-131) 183 201 (GRG . . .) ActRIIb-hFc (R64, 21-131) 121 325 (RGE . . . ) ActRIIb-hFc (R64,22-131) 71 100 (GEA . . . ) ActRIIb-hFc (R64, 23-131) 60 43 (EAE . . . )ActRIIb-hFc (R64, 24-131) 69 105 (AET . . . )

Accordingly, truncations of two, three or four amino acids from theN-terminus lead to the production of a more active protein than theversions with a full-length extracellular domain. Additional experimentsshow that a truncation of five amino acids, ActRIIb-hFc (R64, 25-131)has activity equivalent to the untruncated form, and additionaldeletions at the N-terminus continue to degrade the activity of theprotein. Therefore, optimal constructs will have a C-terminus endingbetween amino acid 133-134 of SEQ ID NO:4 and an N-terminus beginning atamino acids 22-24 of SEQ ID NO:4. An N-terminus corresponding to aminoacids 21 or 25 will give activity that is similar to the ActRIIb-hFc(R64, 20-134) construct.

Example 4. ActRIIb-Fc Variants, Cell-Based Activity

Activity of ActRIIB-Fc proteins was tested in a cell based assay, asdescribed above. Results are summarized in Table 1, below. Some variantswere tested in different C-terminal truncation constructs. As discussedabove, truncations of five or fifteen amino acids caused reduction inactivity. Remarkably, the L79D and L79E variants showed substantial lossof activin binding while retaining almost wild-type inhibition ofGDF-11.

Soluble ActRIIB-Fc Binding to GDF11 and Activin A:

Portion of ActRIIB (corresponds to amino acids GDF11 Activin ActRIIB-Fcof SEQ ID Inhibition Inhibition Variations NO: 4) Activity Activity 64R20-134 +++ +++ (approx. 10⁻⁸M K_(I)) (approx. 10⁻⁸M K_(I)) 64A20-134 + + (approx. 10⁻⁶M K_(I)) (approx. 10⁻⁶M K_(I)) 64R 20-129 ++++++ 64R K74A 20-134 ++++ ++++ 64R A24N 20-134 +++ +++ 64R A24N 20-119 ++++ 64R A24N 20-119 + + K74A R64 L79P 20-134 + + R64 L79P 20-134 + + K74AR64 L79D 20-134 +++ + R64 L79E 20-134 +++ + R64K 20-134 +++ +++ R64K20-129 +++ +++ R64 P129S 20-134 +++ +++ P130A R64N 20-134 + + + Pooractivity (roughly 1 × 10⁻⁶ K_(I)) ++ Moderate activity (roughly 1 × 10⁻⁷K_(I)) +++ Good (wild-type) activity (roughly 1 × 10⁻⁸ K_(I)) ++++Greater than wild-type activity

Several variants have been assessed for serum half-life in rats. ActRIIB(R64 20-134)-Fc has a serum half-life of approximately 70 hours. ActRIIB(R64 A24N 20-134)-Fc has a serum half-life of approximately 100-150hours. The A24N variant has activity in the cell-based assay (above) andin vivo assays (below) that are equivalent to the wild-type molecule.Coupled with the longer half-life, this means that over time an A24Nvariant will give greater effect per unit of protein than the wild-typemolecule.

Remarkably, the introduction of acidic amino acids (aspartic or glutamicacid) at position 79 selectively decreased activin binding whileretaining GDF11/GDF8 binding. As discussed below, wild-type ActRIIB-Fcproteins appear to have effects on tissues other than the muscle, someof which may be undesirable. As disclosed herein, these effects areexpected to relate to the various different ligands that are bound andinhibited by ActRIIB-Fc, including, perhaps, activin. Initial dataindicate that, in mice, the L79E and L79D variants have reduced effectson tissues other than muscle while retaining their effects on muscle.Although variations of this type may be viewed as variants of ActRIIB,it should be noted that these proteins no longer truly function asactivin receptors, and thus the moniker “ActRIIB” is appropriate only asan indicator of the derivation of these polypeptides. Although acidicresidues at position 79 decrease activin binding while retaining GDF11binding, other alterations at this position do not have this effect. AnL79A change increases activin binding relative to GDF11 binding. An L79Pchange decreases both activin and GDF11 binding.

Example 5. GDF-11 and Activin a Binding

Binding of certain ActRIIB-Fc proteins to ligands was tested in aBiaCore™ assay.

The ActRIIB-Fc variants or wild-type protein were captured onto thesystem using an anti-hFc antibody. Ligands were injected and flowed overthe captured receptor proteins. Results are summarized in tables, below.

Ligand Binding Specificity IIB Variants.

GDF11 Protein Kon (1/Ms) Koff (1/s) KD (M) ActRIIB-hFc (R64 20-134)1.34e−6 1.13e−4 8.42e−11 ActRIIB-hFc (R64, A24N 20- 1.21e−6 6.35e−55.19e−11 134) ActRIIB-hFc (R64, L79D 20- 6.7e−5 4.39e−4 6.55e−10 134)ActRIIB-hFc (R64, L79E 20- 3.8e−5 2.74e−4 7.16e−10 134) ActRIIB-hFc(R64K 20-134) 6.77e−5 2.41e−5 3.56e−11

GDF8 Protein Kon (l/Ms) Koff (1/s) KD (M) ActRIIB-hFc (R64 20-134)3.69e−5 3.45e−5 9.35e−11 ActRIIB-hFc (R64, A24N 20- 134) ActRIIB-hFc(R64, L79D 20- 3.85e−5 8.3e−4 2.15e−9 134) ActRIIB-hFc (R64, L79E 20-3.74e−5 9e−4 2.41e−9 134) ActRIIB-hFc (R64K 20-134) 2.25e−5 4.71e−52.1e−10 ActRIIB-hFc (R64K 20-129) 9.74e−4 2.09e−4 2.15e−9 ActRIIB-hFc(R64, P129S, 1.08e−5 1.8e−4 1.67e−9 P130R 20-134) ActRIIB-hFc (R64, K74A20- 2.8e−5 2.03e−5 7.18e−11 134)

ActivinA Protein Kon (l/Ms) Koff (1/s) KD (M) ActRIIB-hFc (R64 20-134)5.94e6 1.59e−4 2.68e−11 ActRIIB-hFc (R64, A24N 20- 3.34e6 3.46e−41.04e−10 134) ActRIIB-hFc (R64, L79D 20- Low binding 134) ActRIIB-hFc(R64, L79E 20- Low binding 134) ActRIIB-hFc (R64K 20-134) 6.82e6 3.25e−44.76e−11 ActRIIB-hFc (R64K 20-129) 7.46e6 6.28e−4 8.41e−11 ActRIIB-hFc(R64, P129S, 5.02e6 4.17e−4 8.31e−11 P130R 20-134)

These data confirm the cell based assay data, demonstrating that theA24N variant retains ligand-binding activity that is similar to that ofthe ActRIIb-hFc (R64 20-134) molecule, and that the L79D or L79Emolecule retains myostatin and GDF11 binding but shows markedlydecreased (non-quantifiable) binding to Activin A.

Other variants have been generated and tested, as reported inWO2006/012627, using ligands coupled to the device and flowing receptorover the coupled ligands. A table of data with respect to these variantsis reproduced below:

Soluble ActRIIB-Fc Variants Binding to GDF11 and Activin a (BiaCoreAssay)

ActRIIB ActA GDF11 WT (64A) KD = 1.8e−7M KD = 2.6e−7M (+) (+) WT (64R)na KD = 8.6e−8M (+++) +15tail KD ~2.6e−8M KD = 1.9e−8M (+++) (++++)E37A * * R40A − − D54A − * K55A ++ * R56A * * K74A KD = 4.35e−9M KD =5.3e−9M +++++ +++++ K74Y * −− K74F * −− K74I * −− W78A * * L79A + *D80K * * D80R * * D80A * * D80F * * D80G * * D80M * * D80N * * D80I * −−F82A ++ − * No observed binding −− <⅕ WT binding − ~ 1/2 WT binding + WT++ <2x increased binding +++ ~5x increased binding ++++ ~10x increasedbinding +++++ ~40x increased binding

Example 6: The Effect of ActRIIB-Fc Proteins on Muscle Mass in Wild-TypeMice

Applicants determined the ability of the ActRIIB-Fc protein to increasemuscle mass in wild-type mice.

C57B110 mice were dosed (10 mg/kg; intraperitoneal (i.p.)) twice/weekwith either the human ActRIIB (R64 20-134) protein or the human ActRIIB(K74A 20-134). Mice were NMR scanned at day 0 and day 28 to determinethe percent change of whole body lean tissue mass. Human ActRIIB (R6420-134)-Fc treated mice exhibited a significant 31.1% increase in leantissue when compared to the vehicle control group. Mice treated with thehuman ActRIIB (K74A 20-134)-Fc protein exhibited a significant increasein lean tissue mass increase compared to the control cohort, albeit to alesser extent than the human ActRIIB (R64 20-134)-treated group. In asimilar study, mice were treated twice/week for with PBS, 1 mg/kg, 3mg/kg, or 10 mg/kg murine ActRIIB (WT, 20-134)-Fc, intraperitoneally. Atthe end of the study, femoris, gastrocnemius, pectoralis and diaphragmmuscles were dissected and weighed. The results are summarized in Table3, below.

TABLE 3 Tissue weights from vehicle-and murine ActRIIB (WT, 20-134)-Fc-treated wild-type mice Gastrocnemius Femoris Pectoralis Dia-Vehicle-treated (L + R) (L + R) (L + R) phragm Average (grams) ± 0.306 ±0.020 0.187 ± 0.040 0.257 ± 0.076 ± Std. deviation 0.020 0.020 muActRIIB(WT, 20- Gastrocnemius Femoris Pectoralis Dia- 134)-Fc (10 mg/kg) (L +R) (L + R) (L + R) phragm Average (grams) ± 0.387 ± 0.010 0.241 ± 0.0140.360 ± 0.124 ± Std. deviation 0.070 0.040 Ttest p-value 0.0001 0.0090.02 0.04As shown in Table 3, the murine ActRIIB (WT, 20-134)-Fc fusion proteinsignificantly increases muscle mass in wild-type mice. In the murineActRIIB (WT, 20-134)-Fc treated mice, gastrocnemius muscles wereincreased 26.5%, femoris muscles increased 28.9%, pectoralis muscleswere increased 40.0%. We also observed changes in the diaphragm musclewhich was increased by 63% compared to the vehicle-treated control mice.The diminution of the diaphragm muscle is a common complication invariety of muscular dystrophies. Therefore the increase in diaphragmweight seen after murine ActRIIB (WT, 20-134)-Fc treatment could be ofclinical importance.

Example 7: The Effect of Long Half-Life ActRIIB-Fc Proteins on MuscleMass in Wild-Type Mice

Applicants determined the ability of the long half-life variant ofActRIIB-mFc (R64, A24N 20-134) protein to increase muscle mass inwild-type mice.

C57B110 mice were dosed (10 mg/kg; intraperitoneal (i.p.)) twice/weekwith either the human ActRIIB-mFc (R64 20-134) protein or the humanActRIIB-mFc (R64, A24N 20-134). Mice were NMR scanned at various pointsup to day 25 to determine the percent change of whole body lean tissuemass. Both molecules caused equivalent increases in total body weightand muscle masses, with the effects on the gastrocnemius, femoris andpectoral muscles ranging from a 40-70% increase. See FIGS. 5 and 6.

These data demonstrate that the increased half-life form of the moleculepromotes muscle growth in a short term study with an equivalent potencyto the wild-type molecule.

Example 8: The Effect of ActRIIB-Fc Proteins with Reduced ActivinBinding on Muscle Mass in Wild-Type Mice

Applicants determined the ability of the long half-life variant ofActRIIB-mFc (R64, L79D 20-134) protein to increase muscle mass inwild-type mice.

C57B110 mice were dosed (10 mg/kg; intraperitoneal (i.p.)) twice/weekwith either the human ActRIIB-mFc (R64 20-134) protein or the humanActRIIB-mFc (R64, L79D 20-134). Mice were NMR scanned at various pointsup to day 24 to determine the percent change of whole body lean tissuemass. Data are shown in the table below.

Body Body Weight Weight Gastrocs Femoris Pecs Day 0 (g) day 24 (g) (L +R) (L + R) (L + R) Mod. TBS 24.4 ± 1.51  26.8 ± 1.43  0.29 ± 0.02 0.17 ±0.24 ± (w/v) 0.02 0.05 R64, 20- 25.0 ± 1.36 31.2* ± 1.53 0.40* ± 0.020.24* ± 0.37* ± 134 (10 0.02 0.07 mg/kg) R64, L79D, 25.3 ± 1.22  28.1 ±1.64 0.32* ± 0.02 0.20* ± 0.27 ± 20-134 0.02 0.05 (10 mg/kg) *p < 0.05

These data demonstrate that the L79D variant (reduced Activin A binding)of ActRIIB is active in vivo for promoting muscle growth, however, theamount of muscle growth is less than that for wild type ActRIIB. Thisdecreased effect may be caused in part by the slight reduction inmyostatin binding or by loss of binding to an additional, as yet unknownnegative regulator of muscle growth. The ability to stimulate musclegrowth without affecting Activin A signaling is highly desirable becauseactivin is a widely expressed regulatory molecule known to have effectson the reproductive system, bone, liver and many other tissues. In mice,ActRIIB-mFc (R64 20-134) causes substantial effects on the reproductivesystem and, in some instances, causes an increase in spleen size. TheActRIIB-mFc (R64, L79D 20-134) molecule had greatly attenuated effectson both reproductive tissues and the spleen, indicating that thismolecule will be particularly suitable for promoting muscle growth inpatients that are reproductively active or have the desire to minimizeeffects on the reproductive system.

Example 9: The Effect of ActRIIB-Fc Protein on Muscle Mass and Strengthin Mdx Mice

In order to determine the ability of the murine ActRIIB (WT, 20-134)-Fcprotein to increase muscle mass in a disease condition, applicantsdetermined the ability of the ActRIIB-Fc protein to increase muscle massin the mdx mouse model of muscular dystrophy.

Adult Mdx mice were treated twice/week with the murine ActRIIB (WT,20-134)-Fc protein (1, 3, or 10 mg/kg; intraperitoneal) or a PBS vehiclecontrol. The force a mouse exerts when pulling a force transducer ismeasured to determine forelimb grip strength. The average force of 5pulling trials was used for the comparison of grip strength between thecohorts. At the end of the study, femoris, gastrocnemius, pectoralis anddiaphragm muscles were dissected and weighed. Grip strength measurementsshowed a significant increase also. The muscle mass results aresummarized in the table, below.

Tissue Weights from Vehicle- and Murine ActRIIB (WT, 20-134)-Fc-TreatedMdx Mice

Gastrocnemius Femoris Pectoralis Dia- Vehicle-treated (L + R) (L + R)(L + R) phragm Average (grams) ± 0.413 ± 0.296 ± 0.437 ± 0.111 ± Std.deviation 0.040 0.019 0.060 0.030 muActRIIB (WT, 20- GastrocnemiusFemoris Pectoralis Dia- 134)-Fc (10 mg/kg) (L + R) (L + R) (L + R)phragm Average (grams) ± 0.52 ± 0.050 0.39 ± 0.05 0.807 ± 0.21 0.149 ±Std. deviation 0.020 Ttest p-value 0.0006 0.0006 0.002 0.05

As illustrated in the table, the murine ActRIIB (WT, 20-134)-Fc-treatedgroups exhibited increased lean tissue mass in the mdx mice compared tothe PBS-treated mice. ActRIIB-Fc treatment increased gastrocnemius size25.9%, femoris size 31.8%, and pectoralis muscles by 85.4% compared tothe vehicle control group. Of possible clinical importance, we alsofound that the diaphragm weights of the mouse ActRIIB (WT,20-134)-Fc-treated mice were increased 34.2% compared to the controlcohort. These data demonstrate the efficacy of the ActRIIB-Fc protein ina muscular dystrophy disease condition.

Additionally mdx mice treated with the ActRIIB-Fc protein exhibitincreased grip strength compared to the vehicle-treated controls. At16-weeks, the 1, 3 and 10 mg/kg ActRIIB groups demonstrated a 31.4%,32.3% and 64.4% increase in grip strength, respectively, compared to thevehicle control group. The improved grip strength performance of themurine ActRIIB (WT, 20-134)-Fc treated groups supports the idea that theincreased muscle found in the treatment groups is physiologicallyrelevant. Mdx mice are susceptible to contractile-induced injury andundergo significantly more cycles of degeneration and regeneration thantheir wild-type counterparts. Despite these muscle phenotypes, murineActRIIB (WT, 20-134)-Fc treatment increases grip strength in the mdxmice.

In Duchenne's Muscular Dystrophy, disease onset occurs early inchildhood, often as early as age five. Accordingly, the data presentedabove with respect to adult mice do not necessarily reflect the effectsan ActRIIB molecule would have in children with DMD. To address this, astudy was conducted with juvenile mdx mice.

ActRIIB-mFc (R64, 20-134) treatment significantly increases body weightin juvenile (four week old) C57BL/10 and mdx mice. Body compositionanalysis using in vivo NMR spectroscopy revealed increased lean tissuemass accompanied the higher body weights. ActRIIB-mFc (R64, 20-134)treated C57BL/10 mice gained 35.2% lean tissue mass and the treated mdxgroup gained 48.3% more lean tissue mass than their respective controlcohorts. Further, the effect of ActRIIB-mFc (R64, 20-134) treatment onstrength was assessed. Vehicle treated mdx mice grip strength scoreswere 15.7% lower than the vehicle C57BL/10 cohort thereby illustratingthe muscle weakness associated with dystrophin deficiency. In contrast,the ActRIIB-mFc (R64, 20-134) treated mdx mice improved their gripstrength compared to the mdx vehicle group, and attained grip strengthmeasurements which surpassed C57BL/10 vehicle mice and reached the levelof the treated C57BL/10 grip strength scores (vehicle mdx: 0.140±0.01KgF; treated mdx: 0.199±0.02 KgF; vehicle C57BL/10: 0.166±0.03;0.205±0.02 KgF). Remarkably, the treatment restored the juvenile mdxmice back to wild type levels of grip strength. Therefore, theActRIIB-mFc (R64, 20-134) molecule is likely to have important clinicalapplications in Duchenne muscular dystrophy, particularly in juvenilepatients at an age close to the onset of the disease.

Example 7: The Effect of ActRIIB-Fc Protein on Strength and Survival inSOD1 Mice

To determine the ability of ActRIIB polypeptides to increase strengthand survival in a mouse model of ALS, applicants tested the ActRIIB-Fcprotein in the SOD1 mouse.

B6.Cg-Tg(SOD1-G93A)1Gur/J, or SOD1, mice carry high copy numbers of themutant allele of the human superoxide dismutase transgene. High levelsof this protein convey a phenotype to the mice that is comparable to thehuman disease ALS. SOD1 mice develop ascending paralysis and exhibitearly signs of the disease by 91 days. The disease results in prematuredeath occurring between 19-23 weeks of age.

SOD1 mice were dosed with a vehicle control or ActRIIB-mFc (K74A 20-134)(i.p., 5 mg/kg, twice/week) beginning at 10 weeks of age. The force amouse exerts when pulling a force transducer is a measure of forelimbgrip strength. The average force of 5 pulling trials was used for thecomparison of grip strength between the cohorts. Survival was calculatedas the number of days between the date the mouse was born and the datethe mouse was unable to right themselves within 30 seconds of beingplaced on its side. FIG. 7 shows the grip strength measurements and FIG.8 illustrates the survival data.

Mice in the end-stage of disease have difficulty grooming, presumablydue to the progression of paralysis, and appear unkempt. Cursoryobservation of the mice revealed that the murine ActRIIB (K74A20-134)-Fc treatment group appeared well-groomed even in the end-stagesof disease compared to the PBS group. This observation suggests thetreated mice are in better health and maintaining a higher quality oflife than the controls.

As is seen in FIG. 7, SOD1 mice receiving the murine ActRIIB (K74A20-134)-Fc treatment exhibit a significantly greater grip strengthcompared to the PBS control cohort. This is seen at day 117, the earlystage of the disease, as well as after the disease has progressed at day149. FIG. 8 illustrates that the ActRIIB (K74A 20-134)-Fc treated micesurvived significantly longer than the vehicle controls. This studyillustrates the utility of the murine ActRIIB (K74A 20-134)-Fc in themouse model of ALS in improving both strength and survival of the mice.

A similar experiment was performed with SOD1 mice, but treatment wasdelayed until the beginning of grossly detectable disease onset (day130), so as to better mimic the treatment of human ALS after onset ofsignificant disease symptoms. At day 130, SOD1 mice were divided intoeither vehicle (modified TBS) or ActRIIB (R64 20-134)-mFc (10 mg/kg)treated groups. Mice were subcutaneously dosed once per week. Mice wereNMR scanned at study days −1 and 27 (ages 129 and 157 days,respectively). Grip strength measurements were performed at study days 0and 20. At the end of the study, the male control group had lost 4.3% oftheir study day 0 body weight whereas the treated group gained 7.8% oftheir study day 0 weights. The female control group lost 1.5% and thetreated female cohort gained 15% of their study day 0 body weights.

SOD1 Grip Strength Measurement

Day 0 Day 20 Male Control 0.149 ± 0.02 0.097 ± 0.02^(a) Male 0.147 ±0.02 0.128 ± 0.02^(a,b) ActRIIB (R64 20- 134)-mFc Female Control 0.130 ±0.02 0.091 ± 0.02^(a) Female 0.128 ± 0.01  0.11 ± 0.02^(b) ActRIIB (R6420- 134)-mFc

Days 0 and 20 grip strength measurements in male and female SOD1 mice.Superscript “a” denotes significantly different compared to therespective day 0 measure (p<0.05). Superscript “b” denotes significantdifference between the PBS (Group 1) and ActRIIB (R64 20-134)-mFc (Group2) day 20 measurements (p<0.05).

Mice were NMR scanned to determine changes in body compositionattributed to treatment. Male control mice lost 6.0% of their leantissue mass over the course of the study (day −1: 18.2 g±1.28; day 27:17.1 g±1.10), Male treated mice gained 9.1% of their study day 0 leantissue mass (day −1: 19.17 g±0.77; day 27: 20.92 g±0.74). Female controlmice had a 0.83% reduction of lean mass from the start of the study (day−1: 13.18 g±0.84; day 27: 13.08 g±0.71) and Female treated mice had a10.7% increase in their study day 0 body weight (day −1: 13.66 g±0.83;day 27: 15.12 g±1.21). Both the male and female treated groups gainedsignificant amounts of lean tissue compared to their respective PBScontrol groups (p<0.001).

SOD1 Muscle Effects of ActRIIB (R64 20-134)-mFc

Gastrocnemius Pectoralis (L + R) Femoris (L + R) (L + R) Male Control0.18 ± 0.03 0.12 ± 0.03 0.20 ± 0.04 Male 0.22 ± 0.04 0.15 ± 0.02 0.30 ±0.04 ActRIIB (R64 20- 134)-mFc Female Control 0.13 ± 0.02 0.089 ± 0.11 ±0.01 0.016 Female 0.17 ± 0.03 0.01 ± 0.02 0.15 ± 0.05 ActRIIB (R64 20-134)-mFc

These data indicated that ActRIIB-Fc treatment may be beneficial in thetreatment of patients that have active ALS, both to improve musclefunction and quality of life.

Example 8: The Effect of an ActRIIB-Fc Protein on the Adiposity andDiabetes in Obese Mice

Applicants tested the ActRIIB-mFc proteins in high fat diet (HFD)-fedmice to determine the ability of ActRIIB-Fc to reduce adiposity in amouse model of obesity.

Type II diabetes is a major complication of obesity and is characterizedby insulin resistance. Elevated fasting insulin levels are indicative ofinsulin resistance and provide a means for testing whether an animal isin an insulin resistant state. Applicants determined the effect oftreatment with murine ActRIIB (R64 K74A 20-134)-Fc in normalizingfasting insulin levels in a mouse model of obesity.

HFD-fed C57BL/6 mice were maintained on a diet composed of 35% fat andconsidered to be obese when their body weight was approximately 50%greater than that of age-matched mice fed a standard chow diet (4.5%fat). Obese mice were dosed twice/week with either a vehicle control orhuman ActRIIB (R64 K74A 20-134)-Fc (10 mg/kg; i.p.). Obese mice were NMRscanned to determine body composition at the beginning of dosing andafter 3 weeks of dosing. The changes in body composition from baselineare summarized in FIG. 9.

Mice were fed a HFD and considered obese when their body weights were50% heavier than their standard chow-fed counterparts. HFD-fed mice weredosed with either a vehicle control or murine ActRIIB (R64 K74A20-134)-Fc (5 mg/kg twice/week; i.p.) for 35 weeks. At the end of thestudy, mice were overnight fasted. At the end of the fast, blood wascollected and processed for serum. Serum was then used to determinefasting insulin levels for both cohorts. The results for the effect ofmurine ActRIIB (K74A 20-134)-Fc on fasting insulin levels of obese miceare summarized in the table, below.

Fasting Insulin Levels from Vehicle- and Murine ActRIIB (K74A20-134)-Fc-Treated Mice

HFD HFD Murine ActRIIB (K74A 20- PBS 134)-mFc Average (ng/ml) ± Std. dev2.27 ± 1.64 0.78 ± 0.40 ttest N/A 0.012

FIG. 9 shows the decreased adiposity of the murine ActRIIB (R64 K74A20-134)-Fc cohort when compared to the vehicle-treated controls. Treatedmice were found to have a 25.9% decrease in fat mass compared to theirbaseline levels. Additionally, the treated group increased their leanmass by 10.1% above their baseline levels. The percent change in boththe adipose tissue and lean tissue mass of the ActRIIB (R64 K74A20-134)-mFc were significantly greater than the percent changes of thePBS-treated group.

In this model, mice were maintained on a high-fat diet until theywere >50% heavier than their chow-fed counterparts. Based on thisremarkable increase in body weight and adiposity, it stands to reasonthat this model could correspond to humans who are characterized asmorbidly obese. Therefore, the finding that treatment with human ActRIIB(R64 K74A 20-134)-Fc protein reduces adiposity in obese mice could beclinically relevant to the treatment of morbidly obese humans.

The results summarized in Table 5 suggest that treatment with the murineActRIIB (K74A 20-134)-Fc protein is able to significantly reduceobesity-associated elevated fasting serum insulin levels. This findingsupports the possible clinical relevance of the use of ActRIIBpolypeptides in the treatment of Type II diabetes.

Further experiments were conducted with ActRIIB-mFc (R64 20-134) in theHFD model of obesity and diabetes. 30 week old HFD-fed C57BL/6 mice weredivided into 2 groups (PBS and 10 mg/kg ActRIIB-mFc (R64 20-134)). Micewere weighed and dosed 2×/week intraperitoneally for 12 weeks. Mice wereassessed by NMR at study days 0 and 94.

Treated mice lost 1.9% of their study day 0 body weights while the PBStreated mice gained 6.7% of their starting BW during the study. Treatedmice also gained significantly more lean tissue than the PBS group(21.1%±6.28 versus 3.7%±4.08) during the study. The Treated mice alsolost significant fat tissue (−34%±10.95) compared to the PBS group(+10.2±10.18).

Individual muscle weights were also increased in the ActRIIB-mFc (R6420-134) treated group.

Gastroc (L + R) Femoris (L + R) Pecs (L + R) PBS 0.33 ± 0.05 0.18 ± 0.030.31 ± 0.05 ActRIIB-mFc 0.44 ± 0.08* 0.25 ± 0.02* 0.44 ± 0.13*(R6420-134) *p < 0.05

In addition to the beneficial effects on fat and muscle that areassociated with ActRIIB-Fc treatment in these mice, positive effects onserum lipids were observed. Both serum cholesterol and triglyceridelevels were markedly reduced, suggesting that ActRIIB-Fc fusion proteinsmay be used to reduce the levels of these lipids in patients.

Example 9: The Effect of ActRIIB-Fc Protein on Muscle Mass in CachecticMice

Applicants tested the ability of ActRIIB (R64 20-134)-mFc to attenuatemuscle loss in a mouse model of glucocorticoid-induced muscle wasting.

Mice were subcutaneously dosed daily for 13 days with either PBS ordexamethasone (2 mg/kg) to induce muscle wasting. Over the same 13 days,PBS- and dexamethosone-treated groups received vehicle or ActRIIB (R6420-134)-mFc (10 mg/kg; i.p.; twice/week) such that all combinations oftreatments were represented. Mice were NMR scanned at days 0 and 13 todetermine changes in lean tissue mass across the groups. NMR results areoutlined in Table 6, below.

TABLE 6 Lean tissue mass of vehicle-and murine ActRIIB (R6420-134)-Fc-treated mice Group Avg lean day 13-Avg lean day 0 (sc:iptreatment) (g) ± std dev PBS:PBS 0.83 ± 0.94 Dexameth:PBS 0.47 ±0.34^(a) Dexameth:ActRIIB 2.56 ± 0.37^(a,b) PBS:ActRIIB 3.63 ± 0.62^(a)^(a)Significant difference compared to PBS:PBS at p < 0.05^(b)Significant difference compared to Dexameth:PBS at p < 0.05

NMR scanning showed a significant 2.5% decrease in lean tissue mass inthe dexamethasone:PBS group compared to the PBS:PBS cohort. In contrast,the dexamethasone:ActRIIB (R64 20-134)-mFc group exhibited a 13.5%increase in lean tissue mass, a significant increase when compared toboth the PBS:PBS and the dexamethasone:PBS groups. Cachexia is anundesirable side effect for a variety of therapeutic treatments,including chronic glucocorticoid therapy. Therefore it could be ofclinical importance that treatment with a human ActRIIB (R64 20-134)-mFcprotein can attenuate the muscle wasting associated with cachexia.

Example 10: The Effect of ActRIIB-Fc on Muscle Mass and Obesity in Agedor Ovariectomized Mice

Sarcopenia is a form of muscle loss associated with aging in otherwisehealthy humans. The disorder is associated with a progressive loss ofskeletal muscle mass and impaired strength and mobility. The causes ofsarcopenia are poorly understood. In women, menopause accelerates muscleloss, much as it does with respect to bone loss. Accordingly, ActRIIB(R64, 20-134)-mFc was tested in extremely old (two year old) mice and inovariectomized mice (a model of the post-menopausal state.

8-week old C57BL/6 female mice were either ovariectomized (OVX) or shamoperated then aged out to 16 weeks before the start of the study. At thebeginning of the study, sham and OVX mice were each divided intotreatment and vehicle groups. All groups were weighed and dosed weeklywith either ActRIIB (R64, 20-134)-mFc or buffer control for 11 weeks.All mice had study days 0 and 83 NMR scans to determine bodycomposition.

At the end of the study, sham PBS mice had lost 4.7% of their originallean mass while the sham treated group increased their lean mass by 21%over the course of the study. OVX controls lost 12.1% (significantlymore than sham vehicle) of their lean mass while treated OVX mice gained12.9% by the end of the study.

These data indicate that ActRIIB-Fc fusion proteins can be used tocounteract the muscle loss that is common in post-menopausal women.

To evaluate the effects of ActRIIB-Fc in a naturally senescentpopulation, male C57BL/6 mice were aged to 70 weeks before the beginningof treatment. Mice were divided into 2 groups (PBS and 10 mg/kg ActRIIB(R64, 20-134)-mFc. Each group was weighed and dosed 2×/week for 10weeks. Over the course of the study, the treated groups gainedsignificantly more lean tissue mass than the PBS group.

% change lean mass PBS 10 mg/kg Average (% from baseline) 101.76 117.27Std dev 3.83 3.91 P-value to PBS <0.001The treated group also had significantly higher individual muscleweights compared to PBS mice.

Muscle Gastoc Femoris Pectoralis weights (L + R) (L + R) (L + R) PBS0.283 ± 0.07 0.156 ± 0.01 0.241 ± 0.07 ActRIIB (R64, 0.371 ± 0.03* 0.192± 0.021* 0.330 ± 0.05* 20-134)-mFc *p < 0.05

Muscle integrity in the treated cohort also appeared to be greater thanthat of the PBS group, as apparently intramuscular fat was reduced andcytoarchitecture improved. (See FIG. 10).

These data demonstrate that ActRIIB-Fc fusion proteins may be used totreat muscle wasting associated with old age in men and women.

Example 11: The Effect of ActRIIB-Fc on Muscle Loss Associated withCastration

Prostate cancer is commonly treated with anti-androgen therapy. Sideeffects of treatment include muscle loss and increased obesity.Castrated mice undergo similar changes, making this a good model for thestudy of the potential for ActRIIB-Fc to be used in this clinicalsetting.

8 week old male C57BL/6 mice were castrated or sham operated thenallowed to recover for 3 weeks before the beginning of the study. Shamand castrated groups were further subdivided into PBS and ActRIIB (R64,20-134)-mFc (10 mg/kg) groups. Mice were weighed and subcutaneouslydosed once/week for 12 weeks. Mice were NMR scanned at study days 0 and83.

Over the course of the study, sham PBS mice gained an average of9.72%±3.67 and sham ActRIIB (R64, 20-134)-mFc mice gained 35.79%±3.1 ofstudy day 0 lean tissue mass. Castrate PBS treated mice lost 8.1%±4.22of their day 0 lean tissue mass while treated castrate mice gained17.77%±3.86. Additionally, castration leads to increased adiposity, butActRIIB (R64, 20-134)-mFc treatment helped to reduce the extent of fatmass gain.

Gastroc and pectoralis muscles from castrated vehicle mice were smallerthan sham PBS mice (castrate gastroc: 0.275±0.03 g, castrate pecs:0.196±0.06 g; sham gastroc: 0.313±0.02 g, sham pecs: 0.254±0.03 g).ActRIIB (R64, 20-134)-mFc treatment significantly attenuates thiscastration-induced decrease in muscle weights (castrate gastroc:0.421±0.03 g, castrate pecs: 0.296±0.06 g).

Example 12: Effects of ActRIIB-Fc on Cancer Cachexia

Many tumors are associated with loss of appetite and severe muscle loss.Patients exhibiting cachexia have a poorer prognosis than non-cachecticpatients. The colon cancer cell line CT26 induces profound cachexia inmice. ActRIIB (R64 20-134) was tested in this model for effects onxenograft-induced cachexia.

Six groups of mice were used in the experiment, as follows:

Group Tumors Treatment Dose Paradigm 1 N VEH v/v Therapuetic 2 NActRIIB-Fc 10 mg/kg Therapeutic 3 Y VEH v/v Therapuetic 4 Y ActRIIB-Fc10 mg/kg Therapeutic 5 Y ActRIIB-Fc 30 mg/kg Therapeutic 6 Y ActRIIB-Fc10 mg/kg PreventativeGroups 3-6 received 5×10{circumflex over ( )}6 tumor cellssubcutaneously. Group 6 began treatment immediately with ActRIIB-Fctwice per week. Groups 1-5 began dosing on study day 28 when tumorsreached a size of 300-500 mm³. As shown in FIG. 11, ActRIIB-Fc markedlydecreased the muscle loss associated with CT26 tumors, both in mice withestablished tumors and when used in a preventative model prior to tumorintroduction.

Example 13: The Effect of ActRIIB-Fc Variants on Muscle Mass inWild-Type Mice

This study showed the effects of the following ActRIIB-related Fcconstructs on muscle mass and other tissues in 6 week old C57BL/6 malemice. Mice were weighed and injected intraperitoneally, biweekly witheither PBS, or an ActRIIB-related Fc constructs (10 mg/kg):

ActRIIB (R64 20-134)-Fc

ActRIIB (L79D 20-134)-Fc

ActRIIB (L79E 20-134)-Fc

ActRIIB (A24N 20-134)-Fc

ActRIIB (R64K 20-134)-Fc

The mice were NMR scanned at the beginning, the middle and the end ofthe study. The femoris, pectoralis and gastrocnemius muscles and theliver, kidneys, and spleen were weighed and saved in formalin.

An initial analysis of the data indicates that ActRIIB (R64 20-134)-Fccauses the greatest increase in muscle mass and lean body mass, whilealso having the greatest effect on other tissues. The L79D and L79Evariants increase muscle mass to a lesser degree, while having littleeffect on other tissues. The A24N and R64K constructs have anintermediate effect on muscle and other tissues. These data confirm thatvariants of ActRIIB with diminished activin binding have desirableproperties, particularly a selective effect on muscle tissue.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference.

While specific embodiments of the subject matter have been discussed,the above specification is illustrative and not restrictive. Manyvariations will become apparent to those skilled in the art upon reviewof this specification and the claims below. The full scope of theinvention should be determined by reference to the claims, along withtheir full scope of equivalents, and the specification, along with suchvariations.

1. A nucleic acid comprising a nucleotide sequence that encodes avariant ActRIIB protein comprising an amino acid sequence that is atleast 80% identical to the sequence of amino acids 29-109 of SEQ ID NO:2, wherein the protein comprises an acidic amino acid at the positioncorresponding to position 79 of SEQ ID NO: 2, and wherein the variantActRIIB protein inhibits signaling by myostatin and/or GDF11 in acell-based assay.
 2. The nucleic acid of claim 1, wherein the variantActRIIB protein comprises an amino acid sequence that is at least 85%,90%, 95%, 97%, or 99% identical to amino acids 29-109 of SEQ ID NO: 2.3. (canceled)
 4. The nucleic acid of claim 1, wherein the variantActRIIB protein comprises at least one N—X—S/T sequence at a positionother than an endogenous N—X—S/T sequence of ActRIIB, and wherein theposition is located outside of the ligand-binding pocket.
 5. The nucleicacid of claim 1, wherein the variant ActRIIB protein comprises an N atthe position corresponding to position 24 of SEQ ID NO: 2 and an S or Tat the position corresponding to position 26 of SEQ ID NO:
 2. 6. Thenucleic acid of claim 1, wherein the variant ActRIIB protein comprisesan R or a K at the position corresponding to position 64 of SEQ ID NO:2.
 7. (canceled)
 8. The nucleic acid of claim 1, wherein the variantActRIIB protein comprises at least one alteration with respect to thesequence of SEQ ID NO: 2 that is a conservative alteration positionedwithin the ligand-binding pocket.
 9. The nucleic acid of claim 1,wherein the variant ActRIIB protein comprises at least one alterationwith respect to the sequence of SEQ ID NO: 2 that is an alteration atone or more positions selected from the group consisting of: K74, R40,Q53, K55, and F82.
 10. The nucleic acid of claim 1, wherein the variantActRIIB protein comprises an amino acid sequence beginning at an aminoacid corresponding to any of amino acids 21-29 of SEQ ID NO: 2 andending at an amino acid corresponding to any of amino acids 128-134 ofSEQ ID NO:
 2. 11. The nucleic acid of claim 1, wherein the variantActRIIB protein is a fusion protein further comprising a heterologousportion.
 12. The nucleic acid of claim 1, wherein the variant ActRIIBprotein is a homodimer.
 13. The nucleic acid of claim 11, wherein theheterologous portion comprises a constant region from an IgG heavychain.
 14. The nucleic acid of claim 11, wherein the heterologousportion comprises an Fc domain.
 15. The nucleic acid of claim 11,wherein the variant ActRIIB protein comprises a linker domain positionedbetween the ActRIIB portion and the heterologous portion.
 16. Thenucleic acid of claim 1, wherein the variant ActRIIB protein comprisesone or more modified amino acid residues selected from: a glycosylatedamino acid, a PEGylated amino acid, a farnesylated amino acid, anacetylated amino acid, a biotinylated amino acid, and an amino acidconjugated to a lipid moiety.
 17. The nucleic acid of claim 1, whereinthe variant ActRIIB protein further comprises a purification sequenceselected from: an epitope tag, a FLAG tag, a polyhistidine sequence, anda GST fusion.
 18. The nucleic acid of claim 1, wherein the amino acid atposition 79 of SEQ ID NO: 2 is a D or an E.
 19. (canceled)
 20. A vectorcomprising the nucleic acid according to claim
 1. 21. A cell comprisingthe vector of claim 20, wherein the cell is not within a human.
 22. Thecell of claim 21, wherein the cell is a CHO cell. 23-25. (canceled)